Multicenter study of the prevalence of carbapenem non-susceptible Enterobacteriaceae

Transcription

1 Multicenter study of the prevalence of carbapenem non-susceptible Enterobacteriaceae (CNSE) and of carbapenemase-producing Enterobacteriaceae (CPE) in Belgium in 2015 Study summary report Study conducted by: 1. National Reference Center (NRC) for Antibiotic-resistant Gram-negative bacilli, CHU Dinant-Godinne UCL Namur (Y. Glupczynski, T.D. Huang) and Hôpital Erasme ULB Brussels (O. Denis), Belgium. 2. A National Multicenter Study Group (see appendix: participating centers and investigators) Version 25/05/2016 1

2 1. Introduction Carbapenemases have been reported extensively worldwide in non-fermenting Gram-negative bacilli (mainly in Pseudomonas spp.) and in Enterobacteriaceae. Colonization or infections 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. The national surveillance program conducted by the national reference center (NRC) for multidrug-resistant Enterobacteriaceae and the Scientific Institute of Public Health (WIV-ISP) has highlighted a dramatic increase since year 2010 of the number of carbapenemase-producing Enterobacteriaceae (CPE) isolates reported in Belgian hospitals. The last multicentric survey performed among 24 Belgian hospitals in 2012 showed an overall point prevalence of 3.5% of carbapenem non-susceptible Enterobacteriaceae (CNSE) and an estimated prevalence of 0.28% of CPE isolates. Klebsiella pneumoniae accounted as the most frequent CPE species and OXA-48 the most frequent carbapenemase enzyme detected. Further, one third of the participating laboratories had isolated one or several CPE isolates in their institution during the study period (Huang et al. JAC 2013). 2. Objectives 1. To screen consecutive non-duplicate Enterobacteriaceae isolates originating from clinical specimens of hospitalized patients for decreased susceptibility to at least one carbapenem drug (ertapenem and meropenem) and the production of carbapenemase. 2. To estimate a point prevalence (over a 2-month period in 2015) of CNSE and of CPE isolates in Belgian hospitals, and to compare the results with those obtained in the 2012 survey. 3. To gain insight of the epidemiology of CNSE and of CPE outside hospitals in specimens collected from community outpatients sent to private laboratories. 3. Materials and methods 3.1 Centers involved (cf. list of participants in the Appendix): Isolates originated from 24 hospital-based laboratories serving medium-to-large size general and tertiary care hospitals across Belgium. For comparison purposes, participants in 2015 were the same as those who already participated to the survey in In addition, 10 private non-hospital based laboratories serving general practitionners and mostly analyzing clinical samples from outpatients in the community were also invited to participate to the present survey. 3.2 Inclusion criteria and testing at participating centers: Isolates to screen for carbapenem susceptibility Version 25/05/2016 2

3 Each laboratory was requested to collect consecutively 200 Enterobacteriaceae isolates (over a maximum period of 2 months): For hospital-based laboratories all clinical specimens and sampling sites collected from hospitalized patients were accepted. On the other hand, screening samples for asymptomatic carriage of multi-drug resistant organisms (stool/rectal swabs) were excluded owing to the large variations of screening strategies between institutions. For private laboratories all collected specimens from all patients were accepted. Only the first isolate of the same species per patient was included (inclusion of duplicates not allowed). Sample collection, culture 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 standardized common protocol: A 0.5 MacFarland density-adjusted suspension of colonies from pure culture was inoculated onto Mueller-Hinton II (MH) agar plates (BioRad). Paper discs (BioRad) of 10-µg meropenem (MEM10) and of 10-µg ertapenem (ETP10) were placed onto the MH plate incubated for 18 hours before reading. All provided paper discs and MH plates for the testing were purchased from the same manufacturer and had the same batch number. For each tested isolate, inhibition zone diameters of the two carbapenems, as well as the sample data were recorded on a standard registration form Isolates to select for characterization of resistance mechanisms Isolates with a decreased carbapenem susceptibility (inhibition zone diameters of ETP10 <25 mm and/or MEM10 <25 mm) according to EUCAST screening breakpoints (EUCAST guidelines for detection of resistance mechanisms v1.0; ) were retained for further analysis. All selected isolates defined as per the above mentioned criteria were first tested for the presence of OXA-48-like and of KPC carbapenemase by OXA-48 K-SeT and by KPC K-SeT immunochromatographic assays (ICT; Coris BioConcept) according to manufacturers instructions. Results of these tests were recorded in the registration form and referred to the reference laboratory together with all retained isolates Quality control (QC) tests Each laboratory was requested to test the E. coli ATCC isolate and two provided carbapenemase-positive (one KPC-2-producing K. pneumoniae NCTC and one OXA-48-producing K. pneumoniae NCTC 13442) at separate intervals of times (at least twice during the survey) for the susceptibility to carbapenems and at least once by the two ICT tests (OXA-48 K-SeT and KPC K- SeT ). Version 25/05/2016 3

4 3.3 Characterization of resistance mechanisms at the reference lab and data analysis: All putative CPE isolates (PCPE) isolates (as defined in 3.2.2) sent to the reference laboratory were retested for in vitro susceptibility by disk diffusion method against a panel of 16 antimicrobial agents, submitted to the electrochemical carbapenemhydrolysis BYG test (Bogaerts et al. JAC 2016) and by in house multiplex end point PCR targeting blavim, blaimp, blandm, blakpc and blaoxa-48 (Bogaerts et al. JAC 2013) for the detection and characterization of carbapenemase encoding genes. Susceptibility rates for the two carbapenems screened, prevalence of CNSE (not susceptible to ertapenem and/or meropenem according to 2015 CLSI criteria which were the same cut-offs used in 2012) and of CPE isolates were calculated overall and individually for each center. Following data analysis using similar microbiological criteria, results from 2015 were compared to those in 2012 (for laboratory-based hospitals only). Version 25/05/2016 4

5 4. Results in hospitals 4.1 Bacterial isolates Overall, 4705 Enterobacteriaceae isolates were screened for decreased carbapenem susceptibility by the 24 participating laboratories from September to November The total number of isolates tested and the proportions of CNSE isolates per center are shown in Table 1. A median number of 200 isolates per laboratory was achieved, 23 centers reached >195 isolates screened and only one center screened 109 isolates (center C15). Overall, 73 isolates were categorized as CNSE using CLSI susceptibility breakpoints representing a point prevalence of clinical CNSE isolates of 1.55% (95% CI 1.20% %) ranging per center from 0% to 3.5%. Compared to 2012 (2.3%; 95% CI 1.9%-2.7%), the overall clinical CNSE prevalence rate decreased significantly (p=0.01 by Pearson Chi-square test). Table 1. Distribution per center of Enterobacteriaceae isolates screened and of CNSE isolates detected (n isolates) Study year Center number Screened CNSE %CNSE 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 total number of isolates screened and of CNSE by sample collection sites, medical wards of the patients at the time of sampling and presumptive time of Version 25/05/2016 5

6 acquisition of the isolate (within 48 h versus after more than 48 h of hospitalization) are shown in Table 2. Compared to 2012, similar distributions of screened isolates were observed in While urine and medical wards represented the principal origins of the isolates screened, the highest proportion of CNSE isolates was found in respiratory tract (2.2%) and in the ICU (2.3%). Table 2. Total number of isolates screened and distribution/proportion of CNSE isolates per sample site, hospitalization unit and per presumptive setting of acquisition (n isolates) Study year Sample data Screened CNSE %CNSE Screened CNSE %CNSE Sample origin Urine % % Respiratory % % Blood % % Pus % % Other % % Ward Medecine % % Surgery % % ICU % % Pediatrics % % Other % % Unknown % 2 0.0% Nosocomial Yes % % No % % Unknown 4 0.0% % Total Total % % The number of tested isolates per species and their susceptibility results to carbapenems (ertapenem and/or meropenem) interpreted according to the 2015 CLSI susceptibility breakpoints are detailed in Table 3. Enterobacter spp. remained the bacterial group displaying the highest proportion of CNSE. However the carbapenem non-susceptibility rate of this group had decreased significantly in comparison to 2012 (5.4% versus 16.7%; p<0.001) and contributed to the overall decrease of the CNSE rate in While the proportion of Enterobacter spp. screened were identical between the two study periods (9%), the CNSE rates among E. cloacae and of E. aerogenes (the two main Enterobacter species) dropped from 16% and 13% to 9% and 1% respectively. K. pneumoniae had become in 2015 the main species (29/73) among CNSE. Table 3. Total number of isolates screened and distribution of CNSE isolates per species or group (n isolates) Study year Species or group Screened Ertapenem I/R Meropenem I/R CNSE %CNSE Screened CNSE %CNSE E. coli % % K. pneumoniae % % K. oxytoca % % Citrobacter spp % % Enterobacter spp % % Proteaceae % % Others % % Total % % Version 25/05/2016 6

7 4.2 Putative CPE isolates referred to the reference laboratory A total of 133 isolates (2.8%) were detected as putative CPE (PCPE) using EUCAST screening breakpoints. Of the 133 PCPE isolates detected at the local laboratories, 114 (86%) were referred centrally for confirmation of carbapenemase production. The distribution of the proportion of CPE by center is listed in Table 4. The minimal estimated proportion of CPE isolates representing the point prevalence of clinical CPE isolates was 0.55% (26/4705; 95% CI 0.34% %) overall and ranged per center from 0% (for 11 centers) to 2.5%. Compared to 2012 (0.25%; 95% CI 0.10%- 0.39%), the overall clinical CPE prevalence rate increased significantly (p=0.02 by Pearson Chi-square test). In total, 13 out of the 24 participating centers collected at least one CPE isolate (vs 8/24 in 2012). Table 4. Distribution per center of putative and confirmed CPE detected (n isolates) Year Center PCPE PCPE NDM, number Screened (EUCAST) referred CPE %CPE OXA-48 KPC NDM VIM OXA-48 CPE %CPE C % 0.00% C % % C % 0.00% C % % C % 0.00% C % % C % % C % % C % 0.00% C % % C % % C % % C % % C % % C % 0.00% C % 0.00% C % % C % 0.00% C % % C % % C % 0.00% C % 0.00% C % % C % % Total % % The number of tested PCPE isolates per species and of CPE isolates confirmed by PCR are shown in Figure 1. Enterobacter spp. accounted for nearly half (53/114; 47%) of the PCPE isolates but none of these could be confirmed as CPE. On the other hand, half of the referred K. pneumoniae (18/34; 53%) were subsequently confirmed as CPE (OXA-48 [n=9], KPC-type [n=7] and NDM-type [n=2]). OXA-48 carbapenemase was detected additionally in 3 E. coli including one coproducing NDM enzyme, 2 C. freundii, one C. koseri and one K. oxytoca isolates. One VIM- Version 25/05/2016 7

8 producing K. oxytoca was also detected by PCR. The proportion of CPE isolates per species/group among the total number of isolates tested was highest in K. pneumoniae (18/629; 2.9%), followed by 2%, 0.9% and 0.1% for, Citrobacter spp. (3/150; 2%), K. oxytoca (2/216; 0.9%) and E. coli (3/2560; 0.1%). Figure 1. Species distribution and carbapenemase coding genes detected in PCPE isolates referred at the reference lab (n=112) E. coli K. pneumoniae K. oxytoca Citrobacter spp. Enterobacter spp. Proteaceae n isolates OXA-48 KPC NDM VIM NDM, OXA-48 Negative The distribution of susceptibility to the two carbapenems tested according to the 2015 CLSI susceptibility breakpoints and the type of carbapenemase encoding gene detected in the 114 PCPE isolates referred to the reference laboratory is summarized in Table 5. All 26 confirmed CPE isolates were categorized as intermediatelyresistant or resistant to ertapenem thus none of these CPE would have been missed if the CLSI susceptibility breakpoint for ertapenem disk was applied as screening criteria. However 9/26 (35%) of the CPE (mainly of OXA-48 type) were classified as meropenem-susceptible including one OXA-48-producing E. coli showing a meropenem disk diameter of 27 mm that would be missed if meropenem was the only carbapenem used with EUCAST screening breakpoint applied. Table 5. Distribution per carbapenemase among PCPE of susceptibility to carbapenems according to the 2015 CLSI interpretative criteria (n isolates) NDM, Ertapenem CLSI Meropenem CLSI OXA-48 KPC NDM VIM OXA-48 Negative Total Ertapenem I/R Meropenem I/R Meropenem S Ertapenem S Meropenem I/R 6 6 Meropenem S Total Version 25/05/2016 8

9 5. Results in private laboratories 5.1 Bacterial isolates Overall, 1991 Enterobacteriaceae isolates were screened for decreased carbapenem susceptibility in the 10 participating private laboratories from September to November The total (global) number of isolates tested and the proportions of CNSE isolates detected per center are shown in Table 6. A median number of 200 isolates per laboratory was achieved ranging 191 to 210 isolates screened. A total of 25 isolates from 6 laboratories would be categorized as CNSE using CLSI susceptibility breakpoints representing a point prevalence of clinical CNSE isolates of 1.25% (95% CI 0.76% %) overall ranging per center from 0% to 4%. Table 6. Distribution per center of Enterobacteriaceae isolates screened and of CNSE isolates detected (n isolates) Center number Screened CNSE %CNSE C % C % C % C % C % C % C % C % C % C % Total % The distribution of screened and of CNSE isolates according to the sample collection sites are shown in Table 7. Compared to a more diversified samples types distribution of screened isolates in hospitals, urinary origin (95% of screened isolates) was largely predominant in private laboratories. The large majority (23/25) of the CNSE isolates were therefore isolated from urine. Table 7. Distribution of total screened and of CNSE isolates per sample origin (n isolates) Sample data Screened CNSE %CNSE Sample origin Urine ,2% Respiratory 15 0,0% Pus ,6% Other 15 0,0% Unknown 3 0,0% Total Total ,3% The number of tested isolates per species and their susceptibility results to carbapenems (ertapenem and/or meropenem) interpreted according to the 2015 CLSI susceptibility breakpoints are detailed in Table 8. Compared to the species distribution of screened isolates in hospitals, as expected a higher proportion of E. coli (64% vs 54% in hospitals) and a lower proportion of Enterobacter spp. (4% vs 9% in hospitals) were screened in private laboratories. K. pneumoniae represented Version 25/05/2016 9

10 the predominant species (14/25) among CNSE although Enterobacter spp. was the group of species showing the highest proportion of CNSE similar to the data in hospitals. Table 8. Distribution of total screened and of CNSE isolates per species or group (n isolates) Species or group Screened Ertapenem I/R Meropenem I/R CNSE %CNSE E. coli % K. pneumoniae % K. oxytoca % Citrobacter spp % Enterobacter spp % Proteaceae % Others % Total % 5.2 Isolates selected for suspicion of CPE and referred to the reference laboratory A total of 42 isolates (2.1%) were detected as putative CPE (PCPE) using EUCAST screening breakpoints. Of the 42 PCPE isolates detected at 7 private laboratories, 30 (71%) were referred to the reference laboratory for confirmation of carbapenemase production. Twelve isolates were confirmed as CPE (11 OXA-48 producers (K. pneumoniae (n=8), 1 E. coli (n=1), K. oxytoca (n=1), C. koseri (n=1), C. freundii (n=1)) and one KPC-producing K. pneumoniae detected from 3 out of the 10 participating centers; the distribution of the proportion of CPE by centre is listed in Table 9. The minimal estimated proportion of CPE isolates representing the point prevalence of CPE isolates was 0.60% (12/1991, 95% CI 0.26%-0.94%) overall and ranged per center from 0% (for 7 centers) to 3.1%. Table 9. Distribution per center of carbapenemase-producing Enterobacteriaceae isolates detected (n isolates) Center number Screened PCPE (EUCAST) PCPE referred OXA-48 KPC CPE %CPE C % C % C % C % C % C % C % C % C % C % Total % Version 25/05/

11 The distribution of susceptibility to the two carbapenems tested according to the 2015 CLSI susceptibility breakpoints among the 30 PCPE isolates referred and characterized at the reference laboratory is summarized in Table 10. Interestingly, one OXA-48-positive C. koseri showing large zone diameters to ertapenem (24 mm) and to meropenem (26 mm) would have been missed if the CLSI susceptibility breakpoints were applied as screening criteria or if meropenem was the only carbapenem disk used with EUCAST screening breakpoint applied. Table 10. Distribution per carbapenemase among PCPE of susceptibility to carbapenems according to the 2015 CLSI interpretative criteria (n isolates) Ertapenem CLSI Meropenem CLSI OXA-48 KPC Negative Total Ertapenem I/R Meropenem I/R Meropenem S Ertapenem S Meropenem I/R 1 1 Meropenem S Total Of note, all isolates of the study yielding OXA-48-positive (n=27) or KPC-positive (n=8) results by the ICT OXA-48 test or the ICT KPC test performed by local laboratories were confirmed at the reference lab as CPE producing the correct corresponding enzymes, while no referred isolates showing negative result with both ICT tests (n=104) were identified as OXA-48 or KPC CPE at the reference lab. Therefore the sensitivity, specificity, positive and negative predictive values were of 100% for these two ICT assays (based on the molecular confirmation of carbapenemase production performed on 144 referred of the 172 potential CPE isolates detected in this study). 6. Quality control testing Regarding the quality control testing (QC) for disk diffusion, 29 centers tested at least twice the three control strains as requested in the study protocol and four centers (C06, C15, C19, C31) did not provide any data of QC. Overall 76 measurements of the two carbapenems tested were obtained with the three control strains. In total with the E. coli ATCC reference strain, 8 and 16 (from C02, C05, C10, C11, C12, C14, C16, C23, C24, C26, C27) results were superior to the acceptable upper QC limit for ertapenem and meropenem disks respectively, while 3 and 3 (from C03, C10) results were inferior to the acceptable lower QC limit respectively for these two carbapenems (QC limits from CLSI 2015). These QC results for E. coli ATCC strain were very similar to the out-of-range QC results obtained in 2012 with higher proportion of results that were above the acceptable upper QC limit for the two carbapenems (Table 11). The overall calculated mean values and one standard deviation (SD) of the two carbapenems disk inhibition diameters are summarized in Figure 2. The highest variation of reading results was observed with ertapenem disk to K. pneumoniae NCTC OXA-48-positive strain, having a SD of 2.7 mm and could be explained by the inconsistently resistant colonies growing within the zone of inhibition frequently observed. The calculated mean disk diameters for ertapenem (33.6 mm) and meropenem (32.7 mm) with E. coli ATCC strain were highly consistent to those obtained (33.6 and 33.0 mm) in the 2012 study. Version 25/05/

12 Table 11. Number (%) of QC results inferior or superior to the acceptable range with E. coli ATCC strain for the two carbapenems disks tested Ertapenem 10 µg Meropenem 10 µg Year (measures) Inferior Superior Inferior Superior 2015 (n=76) 3 (4%) 8 (11%) 3 (4%) 16 (21%) 2012 (n=64) 4 (6%) 10 (16%) 2 (3%) 20 (31%) Figure 2. Mean of carbapenem disks inhibition diameters (mm) to the three quality control strains (76 measures) 40,00 35,00 30,00 25,00 20,00 15,00 10,00 5,00,00 E. coli ATCC (no carbapenemase) K. pneumoniae NCTC (OXA-48) K. pneumoniae NCTC (KPC-3) Ertapenem 10 µg 33,632 15,766 6,468 Meropenem 10 µg 32,711 20,779 7,584 Version 25/05/

13 7. Conclusion In this multicentric 2015 survey in hospitals, we found a significant increased prevalence of clinical CPE in hospitals from 2012 (0.25%) to 2015 (0.55%) with an increased proportion of hospitals-based laboratories (13/24 centers vs 8/24 in 2012) with at least one clinical CPE isolate detected. Although the CPE prevalence rate remained globally low, the study data confirmed the steady progression and spread of CPE in Belgian hospitals over the past three years observed in the national surveillance programme (National Surveillance Report of antimicrobial resistance in Belgian hospitals, D/2015/2505/68, WIV-ISP, B. Jans et al.). In parallel, this first multicentric survey in 10 private laboratories highlighted the presence of CNSE and of CPE in an ambulatory setting. Overall we found a point prevalence of 1.3% of CNSE and a calculated (minimal estimated) prevalence of 0.60% of CPE among ambulant patients. The CPE isolates detected clustered in 3/10 private laboratories which are geographically located close to hospitals with endemicity of CPE suggesting circulation and transfer of CPE carriers between the two health-care sectors. However these data from the participating private labs cannot be easily generalized to the entire community in Belgium since the large majority of these private labs are located in the Northern part of the country. The microbiological analysis confirmed the predominance of K. pneumoniae (26/38; 68%) and of OXA-48 enzyme (27/38; 71%) among CPE isolates detected in hospitals or in the community. Half of the CNSE isolates belonged to E. cloacae and E. aerogenes, but none of them could be confirmed as CPE. Ertapenem disk is clearly a more sensitive screening substrate than meropenem disk for the detection of CPE since 10/26 (38%) of OXA-48 CPE were meropenem susceptible according to CLSI susceptibility breakpoint and two OXA-48 CPE would still be missed even if EUCAST screening breakpoint was applied using meropenem disk used alone. On the other hand, ertapenem resistance alone lacks specificity for the detection of CPE mostly in Enterobacter spp. among which carbapenemase is very rarely found (none in this study). In addition the experience from this multicentric study confirmed excellent performance of the two ICT tests for the rapid and easy-to-perform detection of OXA-48 and KPC CPE (accounting for >90% of total CPE in this study) in a routine lab setting. Version 25/05/

14 Appendix List of the 24 hospital-based laboratories participating to the 2012 and 2015 surveys Laboratories Investigator names AZ St-Jan Brugge Dr. Eric Nulens CHR Namur Dr. Maria-Grazia Garrino CHU Brugmann Dr. Georges Mascart / Ph. Biol. Yvette Miendje CHU Dinant-Godinne Dr. Te-Din Huang / Mme Catherine Berhin CHU Sart-Tilman Liège Pr. Pierette Melin / Dr. Cécile Meex CHU St-Pierre Bruxelles Pr. Olivier Vandenberg / Y. Miendje / Dr. Massin Lebitasy Clinique Saint-Elisabeth Namur Ph. Biol. Terry Laurent Cliniques Braine-L alleud Dr. Yves DeGheldre Cliniques St Joseph Charleroi Dr. Bénédicte Lissoir Cliniques St Pierre Ottignies Ph. Biol. Valérie Verbelen Cliniques Sud Luxembourg Arlon Ph. Biol. Jean-Sébastien Goffinet Cliniques Univ. St-Luc Bruxelles Pr. Anne Simon/ Dr. Hector Rodriguez Villalobos CHR Citadelle Liège Ph. Biol. Pierre Gavage Hôpital Erasme Dr. Olivier Denis / Mme Claire Nonhoff OLV Aalst Dr. Kristien Van Vaerenbergh / Dr. An Boel Imeldaziekenhuis Bonheiden Dr. Johan Frans AZ St-Lucas Gent Dr. Anne.-Marie Vandenabeele CHU Tivoli La Louviere Dr. Catherine Potvliege / Mme Anne Pernet UZ Antwerpen Pr. Greet Ieven / Pr. Herman Goossens UZ Brussel Pr. Denis Pierard / Dr. Deborah de Geyter UZ Leuven Pr. Jan Verhaegen / Dr. Veroniek Saegeman UZ RUG Gent Pr. Geert Claeys / Dr. Jerina Boelens ZOL Genk Dr. Guy Coppens / Dr. Niels Graindor Jessa Ziekenhuis Hasselt Dr. Koen Magerman List of the private laboratories participating to the 2015 survey Laboratories Institut de Biologie Clinique - Bruxelles Laboratoire CPG Bruxelles Medina Dendermonde Nuytinck Evergem KLaboRigo Genk CMA Herentals Medisch Labo Bruyland Kortrijk Algemeen Klinisch Labo Lier Laboratoire J. Woestyn Mouscron Medilab Sint-Niklaas Investigator names Ph. Biol. Brigitte Claude Ph. Biol. Colette Jouniaux Ph. Biol. Evelien Vekens / Ph. Biol. Nele De Wolf Dr. Sc. Herman Nuytten Mr. Ludo Mertens Ph. Biol. Martine Thys Dr. Annemie Vandermeersch / Ph. Biol. Gudrun Depourcq Ph. Biol Linda Joosten Dr. Sophie Woestyn Ph. Biol. Dominique Cuigniez / Ph. Biol. Monique Van Hese Version 25/05/