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Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal carbapenemase-producing organisms in intensive care unit patients

Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal... Background: There are growing concerns regarding the spread of carbapenemase-producing organisms (CPOs) among patients in long-term care facilities (LTCFs) and hospitals in South Korea. We have established a screening protocol for the detection of CPOs in high-risk patients upon admission to intensive care units (ICUs). The diagnostic performance of the Xpert Carba-R assay was compared to that of rectal culture for CPO detection in high-risk patients upon ICU admission. Methods: A total of 408 consecutive rectal swabs were obtained from December 2016 to December 2017. CPO screening was performed using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). When a carbapenemase gene was detected, additional rectal swabs were incubated overnight and inoculated on chromID CARBA medium (bioMérieux, Marcy l’Etoile, France). Bacterial carbapenemase genes, including bla , bla , bla , bla , and KPC NDM VIM IMP-1 bla , were confirmed by conventional PCR. The diagnostic performance of the Carba-R assay was ascertained OXA-48 based on the culture results. Results: The prevalence of CPO carriage was 7.4% according to the Carba-R assay and 3.7% according to rectal culture. The median Ct values of IMP-1 and KPC were significantly different (35.2 vs. 26.6, P = 0.0143). The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba-R assay were 100.0% (95% confidence interval [CI], 78.2–100.0), 96.7% (94.4–98.2), 53.6% (40.4–66.4) and 100.0% (99.0–100.0), respectively. Conclusions: We demonstrated the prevalence of CPO carriage in high-risk patients upon ICU admission and evaluated the diagnostic performance of the Carba-R assay. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of rectal CPO in ICU patients. Keywords: Carbapenemase, Surveillance, Xpert Carba-R assay, Gram-negative bacteria, Intensive care unit, Molecular methods, Chromogenic culture * Correspondence: md.seraph@gmail.com; cklee@korea.ac.kr Department of Laboratory Medicine, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 02841, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 2 of 8 Background evaluate the carbapenemase genes bla , bla , bla- KPC NDM The global transmission of carbapenem-resistant Entero- , bla , and bla . Monobacterial suspensions VIM IMP-1 OXA-48 bacteriaceae (CRE) has accelerated and become a serious of KPC-producing Klebsiella pneumoniae, IMP-1-produ- public health threat worldwide [1, 2]. Carbapenemase-pro- cing Pseudomonas aeruginosa, NDM-producing Entero- ducing organisms (CPOs) include carbapenemase-produ- bacter aerogenes, VIM-producing K. pneumoniae, and cing Enterobacteriaceae (CPE) and carbapenemase- OXA-48-producing Escherichia coli in sterile 0.9% saline producing glucose nonfermenting gram-negative bacilli were adjusted to a 0.5 McFarland standard. Then, sterile (CP-NF), both of which threaten human health [3]. In swabs were placed in the inocula subjected to 10-fold di- 4 3 2 Europe, the prevalence of CPE is 1.3 per 10,000 hospital lutions of 2.25 × 10 , 2.25 × 10 , 2.25 × 10 and 2.25 × admissions, and the resistance rate of last-resort antibi- 10 CFU/swab and vortexed with the reagent for 10 s. otics is high [4]. In South Korea, high rates of carbapenem These inocula in 1.7 mL of reagent solution were trans- resistance among glucose nonfermenting gram-negative ferred into the sample chamber in cartridges. The car- bacilli (CR-NF) and the increased prevalence of CRE have tridges were loaded into a GeneXpert IV instrument become serious problems in hospitalized patients [5, 6]. (Cepheid) according to the manufacturer’s instructions. Since November 2010, nationwide sample surveillance has The LoD claimed by the manufacturer was verified as been conducted, and the number of CPE cases reported in the lowest detected concentration of each of the five South Korea has been dramatically increasing every year CPOs. [7]. The transmission of CPE and associated outbreaks To determine the culture LoD, inocula of 10 CPOs 4 3 have become great concerns because of the difficulties were subjected to 10-fold dilutions of 1 × 10 ,1×10 , 2 1 0 associated with their control in both acute care hospitals 1×10 ,1×10 and 1 × 10 CFU/plate and inoculated on and long-term care facilities (LTCFs) [6, 8]. Risk factors chromID CARBA medium (chromID, bioMérieux, associated with CPE include intensive care therapy, Marcy l’Etoile, France); visible colonies were counted hospitalization in the previous six months, hospital acqui- after 16, 24, and 48 h of incubation at 35 °C. These sition and foreign travel in the previous six months [4]. inocula included five KPC-producing organisms (K. We have established a screening protocol for detecting pneumoniae, E. coli, K. oxytoca, Citrobacter koseri, and CPOs in high-risk patients upon admission to intensive E. cloacae), two NDM producers (E. coli and C. care units (ICUs) using real-time PCR and rectal culture freundii), one IMP-1-producing E. asburiae, one VIM- (Fig. 1). To pre-emptively isolate CPE-colonized patients, producing K. pneumoniae and one OXA-48-producing we actively screen for CPOs using the Xpert Carba-R E. coli. assay (Cepheid, Sunnyvale, CA, USA), which is a rapid and easy-to-use method for detecting five common carba- CRE culture, susceptibility testing and PCR for penemase genes (bla , bla , bla , bla ,and carbapenemase genes KPC NDM VIM IMP-1 bla ). In the present study, we aimed to elucidate the When carbapenemase genes were detected with the OXA-48 prevalence of rectal CPO carriage in high-risk patients ad- Carba-R assay, new rectal swabs were requested for mitted to the ICU at a tertiary hospital and to evaluate the culture during the study period. New swabs were performance of this assay for detecting rectal CPOs. incubated overnight in thioglycolate broth and inocu- lated into chromID. When the organisms were recov- Methods ered, identification and antimicrobial susceptibility Study design testing were performed using the Vitek MS (bioMérieux, This study was conducted as a retrospective study. From Manchester, UK) and Vitek 2 (bioMérieux, Hazelwood, December 2016 to December 2017, rectal swabs were re- MO, USA) instruments. Disk diffusion testing was quested for CPO screening from patients admitted to performed with disks containing 10 μg of ertapenem, the ICU at Korea University Medical Center (KUMC), meropenem and imipenem (Oxoid, Basingstoke, UK) on Guro Hospital. Clinical information on the patients was Muller-Hinton agar. Clinical breakpoints and screening collected from electronic medical records. High-risk cutoff values for CROs were based on EUCAST breakpoint patients were defined as patients who had been previ- tables (http://www.eucast.org/fileadmin/src/media/PDFs/ ously hospitalized in another hospital or LTCF during EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables. the three months before admission to KUMC, Guro pdf). Hospital, or patients known as CPE carriers. When CREs were detected, a modified Hodge test and a carbapenemase inhibition test were performed according to Evaluation of the limit of detection (LoD) the Clinical and Laboratory Standards Institute guidelines Before the study period, we evaluated the LoD of the and diagnosis guideline for CPE [9, 10]. Additionally, in- Xpert Carba-R assay and culture for carbapenem-resist- house PCR for five carbapenemase genes and the Xpert ant organisms (CROs). Five CPO isolates were used to Carba-R assay were performed to confirm the CPOs. Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 3 of 8 Fig. 1 Screening protocols for carbapenemase-producing organisms in this study Briefly, bacterial DNA was extracted using a Genedia respectively, corresponding to 225, 22.5, 2,250, 225 and Mycobacteria DNA Prep Kit (Green Cross Medical Science 225 CFU/swab. Regression analysis generated an equa- Corp., Chungbuk, South Korea), and modified multiplex tion for the assumed bacterial concentration based on PCR for five carbapenemase genes was performed accord- the assigned Ct values (Fig. 2a). According to the equa- ing to a previous report [11]. After the study period, for the tion, the Carba-R assay detected Ct values less than 38.0, discrepant results between the Carba-R assay and cultures, corresponding to a bacterial load greater than 46 CFU/ DNA was extracted from the rectal swabs and sent to a la- swab. boratory (Macrogen, Seoul, South Korea) for bidirectional The LoD of chromID for KPC-producing bacteria sequencing analysis of the five target genes. The sequencing ranged from 1 to 10 CFU/plate, whereas the LoD for primers were designed to include the target regions of the metallo-β-lactamase (MBL)-producing bacteria ranged Carba-R assay in the product sequence. from 10 to 100 CFU/plate. The LoD of chromID for OXA-48-producing bacteria was 1 × 10 CFU/plate Data analysis and statistics (Table 1). Differences in Ct values between the CPO-recovered and non-CPO-recovered swabs were analyzed using Prevalence of rectal CPO carriage the Mann-Whitney test. The sensitivity, specificity, A total of 408 rectal swabs were analyzed during the positive predictive value (PPV) and negative predictive study period. The prevalence of rectal CPO carriage value (NPV) were also calculated. Receiver operating upon admission to the ICU was 7.4% (30 of 408) and characteristic (ROC) curve analysis and diagnostic 3.7% (15 of 406) among high-risk patients according to performance analysis were performed using MedCalc the Carba-R assay and culture results, respectively. IMP- 14.23 (MedCalc Software, Ostend, Belgium). 1 (13, 3.2%) and KPC (10, 2.5%) were predominantly de- tected by the Carba-R assay, followed by NDM (4, 1.0%), Results VIM (0.2%), KPC with OXA-48 (0.2%) and KPC with Evaluation of the LoD for the Carba-R assay and CRE culture IMP-1 (0.2%) (Table 2). The highest Ct values measured for the five CPE isolates Seventeen CPO strains were recovered from newly were 36.5, 37.7, 32.2, 36.9 and 37.0 for the detection of collected 29 rectal swabs. Twelve KPC-producing bla , bla , bla , bla and bla , organisms were recovered from 11 KPC-positive KPC NDM VIM IMP-1 OXA-48 Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 4 of 8 Fig. 2 LoD of the Carba-R assay and comparison of the Ct values combined with CRE cultures. Regression analysis of the Ct values from the 4 3 2 1 Carba-R assay corresponding to bacterial concentrations of 10 ,10 ,10 and 10 CFU/swab (a). The gray line indicates a Ct value of 35 cycles. Comparison of the Ct values from KPC-positive and IMP-1-positive rectal swabs (b). Comparison of the Ct values from rectal swabs with and without CPO recovery (c) rectal swabs, including 7 K. pneumoniae strains, four of less than 390 CFU/swab, and most of these swabs E. coli strains, and one C. koseri strain. One KPC- did not produce CRE culture growth during the study producing K. pneumoniae and one KPC-producing E. period (Fig. 2). The median Ct values of the Carba-R coli strain were recovered from KPC-negative/other assay were significantly different between KPC- and gene-positive rectal swabs. Among the four NDM- IMP-1-producing organisms (26.6 vs. 35.2, P = 0.0143) positive rectal swabs, one NDM-producing K. pneu- (Fig. 2b). Additionally, the median Ct values of the moniae strain was recovered. For most IMP-1-positive assay were significantly different between rectal swabs rectal swabs, the recovered bacteria were not CPOs, containing CPOs and non-CPO rectal swabs (25.9 vs. except two IMP-1-producing P. aeruginosa strains. 36.7, P = 0.0001) (Fig. 2c). VIM- or OXA-48-producing organisms were not re- The overall sensitivity, specificity, PPV and NPV were covered during the study period (Table 2). KPC pro- 100.0, 96.7, 53.6, and 100.0%, respectively, when the Ct ducers (12, 3.0%) were predominantly recovered, followed value recommended by the manufacturer (38.0 cycles) was by IMP-1 producers (2, 0.5%) and NDM producer (1, applied as the cutoff (Table 3). Because of the differences 0.2%). in prevalence among carbapenemase genes, the diagnostic performance varied according to the type of carbapene- Diagnostic performance of the Carba-R assay mase gene detected. Due to the small number of detected In the Carba-R assay, Ct values higher than 35.0 for organisms, the diagnostic performance of VIM and OXA- rectal swabs corresponded to bacterial concentrations 48 in terms of sensitivity and PPV could not be calculated. Table 1 The detection limit of CRE culture stratified by carbapenemase genes Carbapenemases Strains Disk diffusion (mm) The lowest detection limit of CRE culture (CFU/plate) Ertapenem Imipenem Meropenem Ambler class A carbapenemase KPC C. koseri 150 15 18 18 1 KPC E. cloacae 207 7 19 15 10 KPC K. oxytoca 211 18 22 23 1 KPC K. pneumoniae 222 11 17 13 1 KPC E. coli 223 15 17 20 1 MBL carbapenemase NDM C. freundii 148 18 20 19 100 NDM E. coli 192 6 12 10 10 VIM K. pneumoniae 225 18 19 19 10 IMP-1 E. asburiae 44 16 21 19 100 Ambler class D carbapenemase OXA-48 E. coli 229 19 22 23 1 × 10 Abbreviations: MBL metallo-β-lactamase Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 5 of 8 Table 2 Distribution of carbapenemase genes detected by the Xpert Carba-R assay and the recovered carbapenemase-producing organisms Carbapenemase genes (No. of positive Recovered strains from PCR confirmation No. of patients No. of CPO strains results from the Carba-R assay) additional rectal culture of the CPO strains bla (n = 10) Klebsiella pneumoniae KPC CPE 5 5 KPC Escherichia coli KPC CPE 2 2 Klebsiella pneumoniae KPC CPE 1 2 Escherichia coli KPC No growth No growth No CPO 1 0 Not available NA NA 1 0 bla , bla (n =1) Escherichia coli KPC CPE 1 2 KPC IMP-1 Citrobacter koseri KPC bla , bla (n =1) Klebsiella pneumoniae KPC CPE 1 1 KPC OXA-48 Pseudomonas aeruginosa Negative bla (n =4) Escherichia coli KPC CPE 1 1 NDM Klebsiella pneumoniae NDM CPE 1 1 Pseudomonas aeruginosa Negative No CPO 1 0 Klebsiella pneumoniae Negative No CPO 1 0 bla (n =1) Klebsiella pneumoniae KPC CPE 1 1 VIM Escherichia coli Negative bla (n = 13) Pseudomonas aeruginosa IMP-1 CP-NF 2 2 IMP-1 Acinetobacter baumannii Negative No CPO 4 0 Pseudomonas aeruginosa Negative No CPO 2 0 Pseudomonas aeruginosa NA NA 1 0 No growth No growth No CPO 4 0 Abbreviations: CPO carbapenemase-producing organism, CPE carbapenemase-producing Enterobacteriaceae, CP-NF carbapenemase-producing glucose nonfermenting gram-negative bacilli, NA not available The overall specificity and PPV were improved in the Discussion ROC curve analysis when a diagnostic cutoff value of We actively screened for rectal CPOs to support the 35.0 cycles was applied (Table 4). The areas under the pre-emptive isolation of ICU patients, with the results ROC curve (AUCs) were not significantly different in showing that the prevalence of rectal CPOs in high-risk a pairwise comparison of ROC curves when 38.0 and patients using the Carba-R assay was similar to that of a 35.0 cycles were used as cutoff values (0.98 vs. 0.96, previous report in South Korea [5]. The prevalence of P = 0.5405). Using a cutoff value of 35.0 cycles, the rectal CPO carriage varied by region and was observed in overall specificity/PPV improved from 96.7%/53.6% to 0.1% of UK hospitals in 2015 and 19.2% of Spanish hospi- 99.2%/82.4%, with minimal differences in the AUC. tals in 2017 [12, 13]. The distribution of carbapenemase Table 3 Diagnostic performance of the Carba-R assay stratified by carbapenemase gene Carbapenemase gene (No. of CPO carriage No. of specimens with % (95% confidence interval) carbapenemase genes detected by culture (%) the following results by the Carba-R assay) Total TP FP TN* FN Sensitivity Specificity PPV NPV KPC (n = 11)**† 3.0 406 10 1 393 2 83.3 (51.6–97.9) 99.8 (98.6–100.0) 90.9 (58.2–98.6) 99.5 (98.2–99.9) NDM (n = 4) 0.2 406 1 3 402 0 100.0 (2.5–100.0) 99.3 (97.9–99.8) 21.3 (8.1–45.5) 100.0 (99.1–100.0) VIM (n = 1) NA 406 0 1 405 0 NA 99.8 (98.6–100.0) NA 100.0 (99.1–100.0) IMP-1 (n = 13)**† 0.5 406 2 11 393 0 100.0 (15.8–100.0) 97.3 (95.2–98.6) 15.6 (9.3–24.8) 100.0 (99.1–100.0) OXA-48 (n = 1)** NA 406 0 1 405 0 NA 99.8 (98.6–100.0) NA 100.0 (99.1–100.0) Total (n = 28) 3.7 406 15 13 378 0 100.0 (78.2–100.0) 96.7 (94.4–98.2) 53.6 (40.4–66.4) 100.0 (99.0–100.0) *Specimens not detected by the Carba-R assay were considered as true negative results **Two specimens were detected as KPC + IMP-1 and KPC + OXA-48 †The other two specimens were excluded because the results of PCR or CRE culture were not available. Abbreviations: CPO carbapenemase-producing organism, TP true positive, FP false positive, TN true negative, FN false negative, PPV positive predictive value, NPV negative predictive value Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 6 of 8 Table 4 ROC curve analysis of the Carba-R assay stratified by carbapenemase gene and the cutoff Ct value Carbapenemase Cutoff Ct % (95% confidence interval) AUC gene value (cycles) Sensitivity Specificity PPV NPV KPC ≤38.0 83.3 (51.6–97.9) 99.8 (98.6–100.0) 90.9 (58.7–99.8) 99.5 (98.2–99.9) 0.92 (0.88–0.94)* ≤35.0 83.3 (51.6–97.9) 100.0 (99.1–100.0) 100.0 (69.2–100.0) 99.5 (98.2–99.9) 0.92 (0.89–0.94)* IMP-1 ≤38.0 100.0 (15.8–100.0) 97.5 (95.5–98.8) 16.7 (2.1–48.4) 100.0 (99.1–100.0) 0.99 (0.97–1.00)* ≤35.0 50.0 (1.3–98.7) 99.3 (97.8–99.8) 25.0 (0.6–80.6) 99.8 (98.6–100.0) 0.75 (0.70–0.79) Total ≤38.0 100.0 (78.2–100.0) 96.7 (94.4–98.2) 53.6 (33.9–72.5) 100.0 (99.0–100.0) 0.98 (0.97–0.99)* ≤35.0 93.33 (68.1–99.8) 99.2 (97.8–99.8) 82.4 (56.6–96.2) 99.7 (98.6–100.0) 0.96 (0.94–0.98)* *P < 0.0001, Abbreviations: PPV positive predictive value, NPV negative predictive value, AUC area under the ROC curve also exhibited regional variance. In Romania, Spain, and concentration of P. aeruginosa can be considered a Turkey, OXA-48 was most frequently detected among K. major cause of inconsistency with culture results. The pneumoniae, while KPC was the most detected carbape- Carba-R assay can detect the IMP-1, IMP-3, IMP-6, nemase in South Korea, Greece, Italy, and Portugal [4, 6]. IMP-10, IMP-25, and IMP-30 subgroups without dis- The Ct values of the Carba-R assay in this study could tinction. Notably, IMP-6-producing P. aeruginosa has predict CPO recovery in culture. The cutoff Ct value become a predominant clone, and IMP-10-producing was previously reported to distinguish false-positive or P. aeruginosa has recently emerged in South Korea insignificant results from true CPO results [14]. The cut- [17, 18]. In addition, most of the carbapenemase-produ- off value for CPO recovery in this study was 35.0 cycles, cing P. aeruginosa isolates were previously observed to ex- which corresponded to a bacterial concentration of 390 hibit extensive drug resistance and showed susceptibility CFU/swab. The concentration of bla in most speci- only to colistin in 2015 [18]. IMP-1 mens was assumed to be less than 390 CFU/swab in this We found four NDM-positive cases, from which only study. However, the sensitivity of IMP-1 decreased from one NDM-producing bacterial strain was cultured. To ad- 100.0 to 50.0% when using the adjusted cutoff value dress this discrepancy, three NDM- and three IMP-1-posi- (35.0), which resulted from the recovery of one IMP-1 tive/culture-negative specimens were collected, and direct positive specimen with a Ct value of 35.1. Therefore, fur- PCR sequencing of the five carbapenemase genes was per- ther studies are needed since there was an insufficient formed. We detected bla sequences in one NDM- NDM number of recovered IMP-1 producers to determine the positive swab, from which meropenem-susceptible K. recovery cutoff. pneumoniae was recovered. This isolate showed weak The type of carbapenemase gene may affect the CPO positivity in the Hodge test, resistance to ertapenem, and recovery of chromID CARBA medium (chromID). Our susceptibility to imipenem. We also found a bla se- IMP-1 LoD values for chromID ranged from 1 to 10 CFU/plate quence in one IMP-1-positive swab from which bacteria for KPC producers, 10 to 100 CFU/plate for MBL were not recovered. This sequence showed 99.1% carbapenemase producers, and the LoD for OXA-48 homology with bla , bla , bla and bla . IMP-1 IMP-14 IMP-48 IMP-54 producers was 1 × 10 CFU/plate. These results are No other carbapenemase gene sequences were found in consistent with a previous report in which the LoD for the other specimens. In summary, the low concentration KPC-producing bacteria was 1 × 10 CFU/plate, with of CPOs in rectal swabs, the false-positive results obtained 1 6 values of 1 × 10 to 1 × 10 CFU/plate for NDM- and for NDM and IMP-1 using the Carba-R assay and the VIM-producing bacteria and 1 × 10 CFU/plate for weak hydrolysis of MBL carbapenemase were the primary OXA-48-producing bacteria [15]. ChromID is known to causes of discordance between the Carba-R assay and be insufficient for detecting OXA-48-producing bacteria culture results in this study. because of its low analytic sensitivity [14]. However, we Most studies have validated and evaluated the Carba-R did not use ChromID OXA-48 medium during the study assay using contrived specimens of bla producers IMP-1 period due to the low incidence of OXA-48-producing or pure colonies [19–22]. All five types of carbapene- strains in our hospital. mase genes, including bla , were detected with a low IMP-1 We frequently detected the bla gene in this study, prevalence in this study. In addition, the clinical IMP-1 with a detection rate of 3.2%; however, most of the iso- performance of the Carba-R assay using rectal swabs for lated bacteria did not contain this gene. Only two strains active surveillance was reliable and concordant with that of IMP-1-producing P. aeruginosa were recovered from of a previous report, except for the PPV of the bla IMP-1 13 IMP-1-positive rectal swabs. P. aeruginosa is usually and bla genes [19, 20]. The combined use of the NDM present at lower concentrations than other gram-nega- Carba-R assay and culture was found to be a sensitive tive bacteria in the perianal region [16]. Therefore, a low and specific screening method for CPOs [22]. This Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 7 of 8 combination is advantageous because the molecular Abbreviations AST: Antimicrobial susceptibility test; AUC: Area under the ROC curve; method detects only the target gene and allows false- CFU: Colony forming unit; CPE: Carbapenemase-producing Enterobacteriaceae; positive results, whereas rectal culture can identify CP-NF: Carbapenemase-producing glucose nonfermenting gram-negative which bacteria are carbapenem-resistant but may be less bacilli; CPO: Carbapenemase-producing organism; CR-NF: Carbapenem-resistant glucose nonfermenting gram-negative bacilli; CRO: Carbapenem-resistant sensitive because of the abundance of other enteric bac- organism; Ct: threshold cycle; EUCAST: The European Committee on teria [23]. The Carba-R assay enables the detection of Antimicrobial Susceptibility Testing; FN: False negative; FP: False positive; most carbapenemase genes, including the recently emer- ICU: Intensive care unit; IMP: Imipenemase; KPC: Klebsiella pneumoniae carbapenemase; LTCF: Long-term care facility; MBL: Metallo-β-lactamase; ging bla and bla genes; however, bla OXA-181 OXA-232 GES NA: Not available; NDM: New Delhi metallo-β-lactamase; NPV: Negative cannot be detected by the assay [24]. Thus, regional epi- predictive value; OXA: Oxacillinase; PPV: Positive predictive value; ROC: Receiver demiology should be considered when choosing a operating characteristic; TN: True negative; TP: True positive; VIM: Verona integron-encoded metallo-β-lactamase screening method for CPO detection [22]. To prevent CPO transmission, all ICU patients were Acknowledgements pre-emptively isolated on the day of admission and subse- This study was presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) in Madrid, Spain, on 21-24 quently released from quarantine according to the Carba- April 2018. R assay result, which was reported daily. For the patients who tested positive by the Carba-R assay, the quarantine Authors’ contributions YJK devised the study and wrote the manuscript in consultation with CKL. JK was released after three consecutive negative CRE culture and HNK analyzed and interpreted the data. SYY and CSL helped carry out results. This protocol shortened the period of unnecessary the experiments. All authors provided critical feedback and helped shape the patient isolation and reduced medical staff fatigue. research. All authors read and approved the final manuscript. This study has several limitations. We compared the re- Funding sults of the Carba-R assay and CRE cultures for carbapene- This study was supported by a grant from Korea University Medical Center, mase gene-positive specimens. The Carba-R assay was an Guro Hospital (O1801451). imperfect reference method which had the possibility of Availability of data and materials false negativity, especially for a bla gene. After the study KPC Not applicable. period, we compared the Carba-R assay and CRE culture results for 100 PCR-negative specimens. CRE was not cul- Ethics approval and consent to participate This study was approved by the Institutional Review Board (IRB) of the Korea tured for any of the PCR-negative specimens. Among the University Medical Center, Guro Hospital (2018GR0191). The medical records specimens, five strains of carbapenem-susceptible P. aeru- used in this study had been obtained previously as part of routine medical ginosa and one strain of carbapenem-resistant A. bauman- procedures. As the data in this study were collected and analyzed retrospectively, the study did not infringe upon the rights or welfare of the nii complex were recovered. No carbapenemase gene was patients and did not require consent. detected in the strain. The Carba-R assay of prospectively collected rectal swabs from the previous study also did not Consent for publication Not applicable. have a false-negative result [23]. The Carba-R assay did not detect bla , and we could GES Competing interests not exclude the presence of bla in this study. GES The authors declare that they have no competing interests. However, regarding the local epidemiology near the Author details hospital, bla and other carbapenemase genes have GES 1 Department of Laboratory Medicine, Korea University College of Medicine, not been reported [7]. The low prevalence of bla and VIM 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 02841, Republic of Korea. Present address: Department of Laboratory Medicine, Chosun University bla limited the calculation of the sensitivity and OXA-48 Hospital, Gwangju, Republic of Korea. PPV in this study. Further large-scale studies directly comparing culture combined with direct PCR-sequen- Received: 4 April 2019 Accepted: 16 July 2019 cing and the Carba-R assay for CPO detection will eluci- date these limitations. References 1. Albiger B, Glasner C, Struelens MJ, Grundmann H, Monnet DL. Conclusions Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, may 2015. Euro Surveill. 2015;20:1–18. We demonstrated the prevalence of rectal CPO carriage 2. Logan LK, Weinstein RA. The epidemiology of carbapenem-resistant in high-risk patients upon admission to the ICU in a ter- Enterobacteriaceae: the impact and evolution of a global menace. tiary hospital and evaluated the diagnostic performance J Infect Dis. 2017;215:S28–36. 3. Gniadek TJ, Carroll KC, Simner PJ. 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Lin MY, Lyles-Banks RD, Lolans K, Hines DW, Spear JB, Petrak R, et al. The importance of long-term acute care hospitals in the regional epidemiology of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae. Clin Infect Dis. 2013;57:1246–52. 9. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-seventh informational supplement. CLSI document M100-S27. 27th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2017. 10. Song W, Hong SG, Yong D, Jeong SH, Kim HS, Kim HS, et al. Combined use of the modified Hodge test and carbapenemase inhibition test for detection of carbapenemase-producing Enterobacteriaceae and metallo-beta-lactamase-producing pseudomonas spp. Ann Lab Med. 2015;35(2):212–9. 11. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011;70:119–23. 12. Simner PJ, Martin I, Opene B, Tamma PD, Carroll KC, Milstone AM. Evaluation of multiple methods for detection of gastrointestinal colonization of carbapenem-resistant organisms from rectal swabs. J Clin Microbiol. 2016;54:1664–7. 13. García-Castillo M, Reilly M, Pérez-Viso B, Ruiz-Garbajosa P, Moreno R. Evaluation of the BD MAX check-points CPO assay for rapid detection of carbapenemase-producing organisms in rectal swab specimens from high- risk wards [abstract]. In: the European Congress of Clinical Microbiology and th Infectious Diseases 18 . Madrid: Spain; April 2018. p. 21–4. 14. Lau AF, Fahle GA, Kemp MA, Jassem AN, Dekker JP, Frank KM. Clinical performance of check-direct CPE, a multiplex PCR for direct detection of bla , bla and/or bla , and bla from perirectal swabs. J Clin KPC NDM VIM OXA-48 Microbiol. 2015;53:3729–37. 15. Girlich D, Anglade C, Zambardi G, Nordmann P. Comparative evaluation of a novel chromogenic medium (chromID OXA-48) for detection of OXA-48 producing Enterobacteriaceae. Diagn Microbiol Infect Dis. 2013;77:296–300. 16. Warnke P, Pohl FJP, Kundt G, Podbielski A. Screening for gram-negative bacteria: impact of preanalytical parameters. Sci Rep. 2016;6:30427. 17. Yoo JS, Yang JW, Kim HM, Byeon J, Kim HS, Yoo JI, et al. Dissemination of genetically related IMP-6-producing multidrug- resistant Pseudomonas aeruginosa ST235 in South Korea. Int J Antimicrob Agents. 2012;39:300–4. 18. Hong JS, Yoon E-J, Lee H, Jeong S, Lee K. Clonal dissemination of Pseudomonas aeruginosa sequence type 235 isolates carrying blaIMP-6 and emergence of blaGES-24 and blaIMP-10 on novel genomic islands PAGI-15and-16inSouth Korea. Antimicrob Agents Chemother. 2016;60:7216–23. 19. Tato M, Ruiz-Garbajosa P, Traczewski M, Dodgson A, McEwan A, Humphries R, et al. 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Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal carbapenemase-producing organisms in intensive care unit patients

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Biomedicine; Medical Microbiology; Drug Resistance; Infectious Diseases
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10.1186/s13756-019-0579-2
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Abstract

Background: There are growing concerns regarding the spread of carbapenemase-producing organisms (CPOs) among patients in long-term care facilities (LTCFs) and hospitals in South Korea. We have established a screening protocol for the detection of CPOs in high-risk patients upon admission to intensive care units (ICUs). The diagnostic performance of the Xpert Carba-R assay was compared to that of rectal culture for CPO detection in high-risk patients upon ICU admission. Methods: A total of 408 consecutive rectal swabs were obtained from December 2016 to December 2017. CPO screening was performed using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). When a carbapenemase gene was detected, additional rectal swabs were incubated overnight and inoculated on chromID CARBA medium (bioMérieux, Marcy l’Etoile, France). Bacterial carbapenemase genes, including bla , bla , bla , bla , and KPC NDM VIM IMP-1 bla , were confirmed by conventional PCR. The diagnostic performance of the Carba-R assay was ascertained OXA-48 based on the culture results. Results: The prevalence of CPO carriage was 7.4% according to the Carba-R assay and 3.7% according to rectal culture. The median Ct values of IMP-1 and KPC were significantly different (35.2 vs. 26.6, P = 0.0143). The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba-R assay were 100.0% (95% confidence interval [CI], 78.2–100.0), 96.7% (94.4–98.2), 53.6% (40.4–66.4) and 100.0% (99.0–100.0), respectively. Conclusions: We demonstrated the prevalence of CPO carriage in high-risk patients upon ICU admission and evaluated the diagnostic performance of the Carba-R assay. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of rectal CPO in ICU patients. Keywords: Carbapenemase, Surveillance, Xpert Carba-R assay, Gram-negative bacteria, Intensive care unit, Molecular methods, Chromogenic culture * Correspondence: md.seraph@gmail.com; cklee@korea.ac.kr Department of Laboratory Medicine, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 02841, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 2 of 8 Background evaluate the carbapenemase genes bla , bla , bla- KPC NDM The global transmission of carbapenem-resistant Entero- , bla , and bla . Monobacterial suspensions VIM IMP-1 OXA-48 bacteriaceae (CRE) has accelerated and become a serious of KPC-producing Klebsiella pneumoniae, IMP-1-produ- public health threat worldwide [1, 2]. Carbapenemase-pro- cing Pseudomonas aeruginosa, NDM-producing Entero- ducing organisms (CPOs) include carbapenemase-produ- bacter aerogenes, VIM-producing K. pneumoniae, and cing Enterobacteriaceae (CPE) and carbapenemase- OXA-48-producing Escherichia coli in sterile 0.9% saline producing glucose nonfermenting gram-negative bacilli were adjusted to a 0.5 McFarland standard. Then, sterile (CP-NF), both of which threaten human health [3]. In swabs were placed in the inocula subjected to 10-fold di- 4 3 2 Europe, the prevalence of CPE is 1.3 per 10,000 hospital lutions of 2.25 × 10 , 2.25 × 10 , 2.25 × 10 and 2.25 × admissions, and the resistance rate of last-resort antibi- 10 CFU/swab and vortexed with the reagent for 10 s. otics is high [4]. In South Korea, high rates of carbapenem These inocula in 1.7 mL of reagent solution were trans- resistance among glucose nonfermenting gram-negative ferred into the sample chamber in cartridges. The car- bacilli (CR-NF) and the increased prevalence of CRE have tridges were loaded into a GeneXpert IV instrument become serious problems in hospitalized patients [5, 6]. (Cepheid) according to the manufacturer’s instructions. Since November 2010, nationwide sample surveillance has The LoD claimed by the manufacturer was verified as been conducted, and the number of CPE cases reported in the lowest detected concentration of each of the five South Korea has been dramatically increasing every year CPOs. [7]. The transmission of CPE and associated outbreaks To determine the culture LoD, inocula of 10 CPOs 4 3 have become great concerns because of the difficulties were subjected to 10-fold dilutions of 1 × 10 ,1×10 , 2 1 0 associated with their control in both acute care hospitals 1×10 ,1×10 and 1 × 10 CFU/plate and inoculated on and long-term care facilities (LTCFs) [6, 8]. Risk factors chromID CARBA medium (chromID, bioMérieux, associated with CPE include intensive care therapy, Marcy l’Etoile, France); visible colonies were counted hospitalization in the previous six months, hospital acqui- after 16, 24, and 48 h of incubation at 35 °C. These sition and foreign travel in the previous six months [4]. inocula included five KPC-producing organisms (K. We have established a screening protocol for detecting pneumoniae, E. coli, K. oxytoca, Citrobacter koseri, and CPOs in high-risk patients upon admission to intensive E. cloacae), two NDM producers (E. coli and C. care units (ICUs) using real-time PCR and rectal culture freundii), one IMP-1-producing E. asburiae, one VIM- (Fig. 1). To pre-emptively isolate CPE-colonized patients, producing K. pneumoniae and one OXA-48-producing we actively screen for CPOs using the Xpert Carba-R E. coli. assay (Cepheid, Sunnyvale, CA, USA), which is a rapid and easy-to-use method for detecting five common carba- CRE culture, susceptibility testing and PCR for penemase genes (bla , bla , bla , bla ,and carbapenemase genes KPC NDM VIM IMP-1 bla ). In the present study, we aimed to elucidate the When carbapenemase genes were detected with the OXA-48 prevalence of rectal CPO carriage in high-risk patients ad- Carba-R assay, new rectal swabs were requested for mitted to the ICU at a tertiary hospital and to evaluate the culture during the study period. New swabs were performance of this assay for detecting rectal CPOs. incubated overnight in thioglycolate broth and inocu- lated into chromID. When the organisms were recov- Methods ered, identification and antimicrobial susceptibility Study design testing were performed using the Vitek MS (bioMérieux, This study was conducted as a retrospective study. From Manchester, UK) and Vitek 2 (bioMérieux, Hazelwood, December 2016 to December 2017, rectal swabs were re- MO, USA) instruments. Disk diffusion testing was quested for CPO screening from patients admitted to performed with disks containing 10 μg of ertapenem, the ICU at Korea University Medical Center (KUMC), meropenem and imipenem (Oxoid, Basingstoke, UK) on Guro Hospital. Clinical information on the patients was Muller-Hinton agar. Clinical breakpoints and screening collected from electronic medical records. High-risk cutoff values for CROs were based on EUCAST breakpoint patients were defined as patients who had been previ- tables (http://www.eucast.org/fileadmin/src/media/PDFs/ ously hospitalized in another hospital or LTCF during EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables. the three months before admission to KUMC, Guro pdf). Hospital, or patients known as CPE carriers. When CREs were detected, a modified Hodge test and a carbapenemase inhibition test were performed according to Evaluation of the limit of detection (LoD) the Clinical and Laboratory Standards Institute guidelines Before the study period, we evaluated the LoD of the and diagnosis guideline for CPE [9, 10]. Additionally, in- Xpert Carba-R assay and culture for carbapenem-resist- house PCR for five carbapenemase genes and the Xpert ant organisms (CROs). Five CPO isolates were used to Carba-R assay were performed to confirm the CPOs. Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 3 of 8 Fig. 1 Screening protocols for carbapenemase-producing organisms in this study Briefly, bacterial DNA was extracted using a Genedia respectively, corresponding to 225, 22.5, 2,250, 225 and Mycobacteria DNA Prep Kit (Green Cross Medical Science 225 CFU/swab. Regression analysis generated an equa- Corp., Chungbuk, South Korea), and modified multiplex tion for the assumed bacterial concentration based on PCR for five carbapenemase genes was performed accord- the assigned Ct values (Fig. 2a). According to the equa- ing to a previous report [11]. After the study period, for the tion, the Carba-R assay detected Ct values less than 38.0, discrepant results between the Carba-R assay and cultures, corresponding to a bacterial load greater than 46 CFU/ DNA was extracted from the rectal swabs and sent to a la- swab. boratory (Macrogen, Seoul, South Korea) for bidirectional The LoD of chromID for KPC-producing bacteria sequencing analysis of the five target genes. The sequencing ranged from 1 to 10 CFU/plate, whereas the LoD for primers were designed to include the target regions of the metallo-β-lactamase (MBL)-producing bacteria ranged Carba-R assay in the product sequence. from 10 to 100 CFU/plate. The LoD of chromID for OXA-48-producing bacteria was 1 × 10 CFU/plate Data analysis and statistics (Table 1). Differences in Ct values between the CPO-recovered and non-CPO-recovered swabs were analyzed using Prevalence of rectal CPO carriage the Mann-Whitney test. The sensitivity, specificity, A total of 408 rectal swabs were analyzed during the positive predictive value (PPV) and negative predictive study period. The prevalence of rectal CPO carriage value (NPV) were also calculated. Receiver operating upon admission to the ICU was 7.4% (30 of 408) and characteristic (ROC) curve analysis and diagnostic 3.7% (15 of 406) among high-risk patients according to performance analysis were performed using MedCalc the Carba-R assay and culture results, respectively. IMP- 14.23 (MedCalc Software, Ostend, Belgium). 1 (13, 3.2%) and KPC (10, 2.5%) were predominantly de- tected by the Carba-R assay, followed by NDM (4, 1.0%), Results VIM (0.2%), KPC with OXA-48 (0.2%) and KPC with Evaluation of the LoD for the Carba-R assay and CRE culture IMP-1 (0.2%) (Table 2). The highest Ct values measured for the five CPE isolates Seventeen CPO strains were recovered from newly were 36.5, 37.7, 32.2, 36.9 and 37.0 for the detection of collected 29 rectal swabs. Twelve KPC-producing bla , bla , bla , bla and bla , organisms were recovered from 11 KPC-positive KPC NDM VIM IMP-1 OXA-48 Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 4 of 8 Fig. 2 LoD of the Carba-R assay and comparison of the Ct values combined with CRE cultures. Regression analysis of the Ct values from the 4 3 2 1 Carba-R assay corresponding to bacterial concentrations of 10 ,10 ,10 and 10 CFU/swab (a). The gray line indicates a Ct value of 35 cycles. Comparison of the Ct values from KPC-positive and IMP-1-positive rectal swabs (b). Comparison of the Ct values from rectal swabs with and without CPO recovery (c) rectal swabs, including 7 K. pneumoniae strains, four of less than 390 CFU/swab, and most of these swabs E. coli strains, and one C. koseri strain. One KPC- did not produce CRE culture growth during the study producing K. pneumoniae and one KPC-producing E. period (Fig. 2). The median Ct values of the Carba-R coli strain were recovered from KPC-negative/other assay were significantly different between KPC- and gene-positive rectal swabs. Among the four NDM- IMP-1-producing organisms (26.6 vs. 35.2, P = 0.0143) positive rectal swabs, one NDM-producing K. pneu- (Fig. 2b). Additionally, the median Ct values of the moniae strain was recovered. For most IMP-1-positive assay were significantly different between rectal swabs rectal swabs, the recovered bacteria were not CPOs, containing CPOs and non-CPO rectal swabs (25.9 vs. except two IMP-1-producing P. aeruginosa strains. 36.7, P = 0.0001) (Fig. 2c). VIM- or OXA-48-producing organisms were not re- The overall sensitivity, specificity, PPV and NPV were covered during the study period (Table 2). KPC pro- 100.0, 96.7, 53.6, and 100.0%, respectively, when the Ct ducers (12, 3.0%) were predominantly recovered, followed value recommended by the manufacturer (38.0 cycles) was by IMP-1 producers (2, 0.5%) and NDM producer (1, applied as the cutoff (Table 3). Because of the differences 0.2%). in prevalence among carbapenemase genes, the diagnostic performance varied according to the type of carbapene- Diagnostic performance of the Carba-R assay mase gene detected. Due to the small number of detected In the Carba-R assay, Ct values higher than 35.0 for organisms, the diagnostic performance of VIM and OXA- rectal swabs corresponded to bacterial concentrations 48 in terms of sensitivity and PPV could not be calculated. Table 1 The detection limit of CRE culture stratified by carbapenemase genes Carbapenemases Strains Disk diffusion (mm) The lowest detection limit of CRE culture (CFU/plate) Ertapenem Imipenem Meropenem Ambler class A carbapenemase KPC C. koseri 150 15 18 18 1 KPC E. cloacae 207 7 19 15 10 KPC K. oxytoca 211 18 22 23 1 KPC K. pneumoniae 222 11 17 13 1 KPC E. coli 223 15 17 20 1 MBL carbapenemase NDM C. freundii 148 18 20 19 100 NDM E. coli 192 6 12 10 10 VIM K. pneumoniae 225 18 19 19 10 IMP-1 E. asburiae 44 16 21 19 100 Ambler class D carbapenemase OXA-48 E. coli 229 19 22 23 1 × 10 Abbreviations: MBL metallo-β-lactamase Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 5 of 8 Table 2 Distribution of carbapenemase genes detected by the Xpert Carba-R assay and the recovered carbapenemase-producing organisms Carbapenemase genes (No. of positive Recovered strains from PCR confirmation No. of patients No. of CPO strains results from the Carba-R assay) additional rectal culture of the CPO strains bla (n = 10) Klebsiella pneumoniae KPC CPE 5 5 KPC Escherichia coli KPC CPE 2 2 Klebsiella pneumoniae KPC CPE 1 2 Escherichia coli KPC No growth No growth No CPO 1 0 Not available NA NA 1 0 bla , bla (n =1) Escherichia coli KPC CPE 1 2 KPC IMP-1 Citrobacter koseri KPC bla , bla (n =1) Klebsiella pneumoniae KPC CPE 1 1 KPC OXA-48 Pseudomonas aeruginosa Negative bla (n =4) Escherichia coli KPC CPE 1 1 NDM Klebsiella pneumoniae NDM CPE 1 1 Pseudomonas aeruginosa Negative No CPO 1 0 Klebsiella pneumoniae Negative No CPO 1 0 bla (n =1) Klebsiella pneumoniae KPC CPE 1 1 VIM Escherichia coli Negative bla (n = 13) Pseudomonas aeruginosa IMP-1 CP-NF 2 2 IMP-1 Acinetobacter baumannii Negative No CPO 4 0 Pseudomonas aeruginosa Negative No CPO 2 0 Pseudomonas aeruginosa NA NA 1 0 No growth No growth No CPO 4 0 Abbreviations: CPO carbapenemase-producing organism, CPE carbapenemase-producing Enterobacteriaceae, CP-NF carbapenemase-producing glucose nonfermenting gram-negative bacilli, NA not available The overall specificity and PPV were improved in the Discussion ROC curve analysis when a diagnostic cutoff value of We actively screened for rectal CPOs to support the 35.0 cycles was applied (Table 4). The areas under the pre-emptive isolation of ICU patients, with the results ROC curve (AUCs) were not significantly different in showing that the prevalence of rectal CPOs in high-risk a pairwise comparison of ROC curves when 38.0 and patients using the Carba-R assay was similar to that of a 35.0 cycles were used as cutoff values (0.98 vs. 0.96, previous report in South Korea [5]. The prevalence of P = 0.5405). Using a cutoff value of 35.0 cycles, the rectal CPO carriage varied by region and was observed in overall specificity/PPV improved from 96.7%/53.6% to 0.1% of UK hospitals in 2015 and 19.2% of Spanish hospi- 99.2%/82.4%, with minimal differences in the AUC. tals in 2017 [12, 13]. The distribution of carbapenemase Table 3 Diagnostic performance of the Carba-R assay stratified by carbapenemase gene Carbapenemase gene (No. of CPO carriage No. of specimens with % (95% confidence interval) carbapenemase genes detected by culture (%) the following results by the Carba-R assay) Total TP FP TN* FN Sensitivity Specificity PPV NPV KPC (n = 11)**† 3.0 406 10 1 393 2 83.3 (51.6–97.9) 99.8 (98.6–100.0) 90.9 (58.2–98.6) 99.5 (98.2–99.9) NDM (n = 4) 0.2 406 1 3 402 0 100.0 (2.5–100.0) 99.3 (97.9–99.8) 21.3 (8.1–45.5) 100.0 (99.1–100.0) VIM (n = 1) NA 406 0 1 405 0 NA 99.8 (98.6–100.0) NA 100.0 (99.1–100.0) IMP-1 (n = 13)**† 0.5 406 2 11 393 0 100.0 (15.8–100.0) 97.3 (95.2–98.6) 15.6 (9.3–24.8) 100.0 (99.1–100.0) OXA-48 (n = 1)** NA 406 0 1 405 0 NA 99.8 (98.6–100.0) NA 100.0 (99.1–100.0) Total (n = 28) 3.7 406 15 13 378 0 100.0 (78.2–100.0) 96.7 (94.4–98.2) 53.6 (40.4–66.4) 100.0 (99.0–100.0) *Specimens not detected by the Carba-R assay were considered as true negative results **Two specimens were detected as KPC + IMP-1 and KPC + OXA-48 †The other two specimens were excluded because the results of PCR or CRE culture were not available. Abbreviations: CPO carbapenemase-producing organism, TP true positive, FP false positive, TN true negative, FN false negative, PPV positive predictive value, NPV negative predictive value Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 6 of 8 Table 4 ROC curve analysis of the Carba-R assay stratified by carbapenemase gene and the cutoff Ct value Carbapenemase Cutoff Ct % (95% confidence interval) AUC gene value (cycles) Sensitivity Specificity PPV NPV KPC ≤38.0 83.3 (51.6–97.9) 99.8 (98.6–100.0) 90.9 (58.7–99.8) 99.5 (98.2–99.9) 0.92 (0.88–0.94)* ≤35.0 83.3 (51.6–97.9) 100.0 (99.1–100.0) 100.0 (69.2–100.0) 99.5 (98.2–99.9) 0.92 (0.89–0.94)* IMP-1 ≤38.0 100.0 (15.8–100.0) 97.5 (95.5–98.8) 16.7 (2.1–48.4) 100.0 (99.1–100.0) 0.99 (0.97–1.00)* ≤35.0 50.0 (1.3–98.7) 99.3 (97.8–99.8) 25.0 (0.6–80.6) 99.8 (98.6–100.0) 0.75 (0.70–0.79) Total ≤38.0 100.0 (78.2–100.0) 96.7 (94.4–98.2) 53.6 (33.9–72.5) 100.0 (99.0–100.0) 0.98 (0.97–0.99)* ≤35.0 93.33 (68.1–99.8) 99.2 (97.8–99.8) 82.4 (56.6–96.2) 99.7 (98.6–100.0) 0.96 (0.94–0.98)* *P < 0.0001, Abbreviations: PPV positive predictive value, NPV negative predictive value, AUC area under the ROC curve also exhibited regional variance. In Romania, Spain, and concentration of P. aeruginosa can be considered a Turkey, OXA-48 was most frequently detected among K. major cause of inconsistency with culture results. The pneumoniae, while KPC was the most detected carbape- Carba-R assay can detect the IMP-1, IMP-3, IMP-6, nemase in South Korea, Greece, Italy, and Portugal [4, 6]. IMP-10, IMP-25, and IMP-30 subgroups without dis- The Ct values of the Carba-R assay in this study could tinction. Notably, IMP-6-producing P. aeruginosa has predict CPO recovery in culture. The cutoff Ct value become a predominant clone, and IMP-10-producing was previously reported to distinguish false-positive or P. aeruginosa has recently emerged in South Korea insignificant results from true CPO results [14]. The cut- [17, 18]. In addition, most of the carbapenemase-produ- off value for CPO recovery in this study was 35.0 cycles, cing P. aeruginosa isolates were previously observed to ex- which corresponded to a bacterial concentration of 390 hibit extensive drug resistance and showed susceptibility CFU/swab. The concentration of bla in most speci- only to colistin in 2015 [18]. IMP-1 mens was assumed to be less than 390 CFU/swab in this We found four NDM-positive cases, from which only study. However, the sensitivity of IMP-1 decreased from one NDM-producing bacterial strain was cultured. To ad- 100.0 to 50.0% when using the adjusted cutoff value dress this discrepancy, three NDM- and three IMP-1-posi- (35.0), which resulted from the recovery of one IMP-1 tive/culture-negative specimens were collected, and direct positive specimen with a Ct value of 35.1. Therefore, fur- PCR sequencing of the five carbapenemase genes was per- ther studies are needed since there was an insufficient formed. We detected bla sequences in one NDM- NDM number of recovered IMP-1 producers to determine the positive swab, from which meropenem-susceptible K. recovery cutoff. pneumoniae was recovered. This isolate showed weak The type of carbapenemase gene may affect the CPO positivity in the Hodge test, resistance to ertapenem, and recovery of chromID CARBA medium (chromID). Our susceptibility to imipenem. We also found a bla se- IMP-1 LoD values for chromID ranged from 1 to 10 CFU/plate quence in one IMP-1-positive swab from which bacteria for KPC producers, 10 to 100 CFU/plate for MBL were not recovered. This sequence showed 99.1% carbapenemase producers, and the LoD for OXA-48 homology with bla , bla , bla and bla . IMP-1 IMP-14 IMP-48 IMP-54 producers was 1 × 10 CFU/plate. These results are No other carbapenemase gene sequences were found in consistent with a previous report in which the LoD for the other specimens. In summary, the low concentration KPC-producing bacteria was 1 × 10 CFU/plate, with of CPOs in rectal swabs, the false-positive results obtained 1 6 values of 1 × 10 to 1 × 10 CFU/plate for NDM- and for NDM and IMP-1 using the Carba-R assay and the VIM-producing bacteria and 1 × 10 CFU/plate for weak hydrolysis of MBL carbapenemase were the primary OXA-48-producing bacteria [15]. ChromID is known to causes of discordance between the Carba-R assay and be insufficient for detecting OXA-48-producing bacteria culture results in this study. because of its low analytic sensitivity [14]. However, we Most studies have validated and evaluated the Carba-R did not use ChromID OXA-48 medium during the study assay using contrived specimens of bla producers IMP-1 period due to the low incidence of OXA-48-producing or pure colonies [19–22]. All five types of carbapene- strains in our hospital. mase genes, including bla , were detected with a low IMP-1 We frequently detected the bla gene in this study, prevalence in this study. In addition, the clinical IMP-1 with a detection rate of 3.2%; however, most of the iso- performance of the Carba-R assay using rectal swabs for lated bacteria did not contain this gene. Only two strains active surveillance was reliable and concordant with that of IMP-1-producing P. aeruginosa were recovered from of a previous report, except for the PPV of the bla IMP-1 13 IMP-1-positive rectal swabs. P. aeruginosa is usually and bla genes [19, 20]. The combined use of the NDM present at lower concentrations than other gram-nega- Carba-R assay and culture was found to be a sensitive tive bacteria in the perianal region [16]. Therefore, a low and specific screening method for CPOs [22]. This Ko et al. Antimicrobial Resistance and Infection Control (2019) 8:127 Page 7 of 8 combination is advantageous because the molecular Abbreviations AST: Antimicrobial susceptibility test; AUC: Area under the ROC curve; method detects only the target gene and allows false- CFU: Colony forming unit; CPE: Carbapenemase-producing Enterobacteriaceae; positive results, whereas rectal culture can identify CP-NF: Carbapenemase-producing glucose nonfermenting gram-negative which bacteria are carbapenem-resistant but may be less bacilli; CPO: Carbapenemase-producing organism; CR-NF: Carbapenem-resistant glucose nonfermenting gram-negative bacilli; CRO: Carbapenem-resistant sensitive because of the abundance of other enteric bac- organism; Ct: threshold cycle; EUCAST: The European Committee on teria [23]. The Carba-R assay enables the detection of Antimicrobial Susceptibility Testing; FN: False negative; FP: False positive; most carbapenemase genes, including the recently emer- ICU: Intensive care unit; IMP: Imipenemase; KPC: Klebsiella pneumoniae carbapenemase; LTCF: Long-term care facility; MBL: Metallo-β-lactamase; ging bla and bla genes; however, bla OXA-181 OXA-232 GES NA: Not available; NDM: New Delhi metallo-β-lactamase; NPV: Negative cannot be detected by the assay [24]. Thus, regional epi- predictive value; OXA: Oxacillinase; PPV: Positive predictive value; ROC: Receiver demiology should be considered when choosing a operating characteristic; TN: True negative; TP: True positive; VIM: Verona integron-encoded metallo-β-lactamase screening method for CPO detection [22]. To prevent CPO transmission, all ICU patients were Acknowledgements pre-emptively isolated on the day of admission and subse- This study was presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) in Madrid, Spain, on 21-24 quently released from quarantine according to the Carba- April 2018. R assay result, which was reported daily. For the patients who tested positive by the Carba-R assay, the quarantine Authors’ contributions YJK devised the study and wrote the manuscript in consultation with CKL. JK was released after three consecutive negative CRE culture and HNK analyzed and interpreted the data. SYY and CSL helped carry out results. This protocol shortened the period of unnecessary the experiments. All authors provided critical feedback and helped shape the patient isolation and reduced medical staff fatigue. research. All authors read and approved the final manuscript. This study has several limitations. We compared the re- Funding sults of the Carba-R assay and CRE cultures for carbapene- This study was supported by a grant from Korea University Medical Center, mase gene-positive specimens. The Carba-R assay was an Guro Hospital (O1801451). imperfect reference method which had the possibility of Availability of data and materials false negativity, especially for a bla gene. After the study KPC Not applicable. period, we compared the Carba-R assay and CRE culture results for 100 PCR-negative specimens. CRE was not cul- Ethics approval and consent to participate This study was approved by the Institutional Review Board (IRB) of the Korea tured for any of the PCR-negative specimens. Among the University Medical Center, Guro Hospital (2018GR0191). The medical records specimens, five strains of carbapenem-susceptible P. aeru- used in this study had been obtained previously as part of routine medical ginosa and one strain of carbapenem-resistant A. bauman- procedures. As the data in this study were collected and analyzed retrospectively, the study did not infringe upon the rights or welfare of the nii complex were recovered. No carbapenemase gene was patients and did not require consent. detected in the strain. The Carba-R assay of prospectively collected rectal swabs from the previous study also did not Consent for publication Not applicable. have a false-negative result [23]. The Carba-R assay did not detect bla , and we could GES Competing interests not exclude the presence of bla in this study. GES The authors declare that they have no competing interests. However, regarding the local epidemiology near the Author details hospital, bla and other carbapenemase genes have GES 1 Department of Laboratory Medicine, Korea University College of Medicine, not been reported [7]. The low prevalence of bla and VIM 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 02841, Republic of Korea. Present address: Department of Laboratory Medicine, Chosun University bla limited the calculation of the sensitivity and OXA-48 Hospital, Gwangju, Republic of Korea. PPV in this study. Further large-scale studies directly comparing culture combined with direct PCR-sequen- Received: 4 April 2019 Accepted: 16 July 2019 cing and the Carba-R assay for CPO detection will eluci- date these limitations. References 1. Albiger B, Glasner C, Struelens MJ, Grundmann H, Monnet DL. Conclusions Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, may 2015. Euro Surveill. 2015;20:1–18. We demonstrated the prevalence of rectal CPO carriage 2. Logan LK, Weinstein RA. The epidemiology of carbapenem-resistant in high-risk patients upon admission to the ICU in a ter- Enterobacteriaceae: the impact and evolution of a global menace. tiary hospital and evaluated the diagnostic performance J Infect Dis. 2017;215:S28–36. 3. Gniadek TJ, Carroll KC, Simner PJ. 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Antimicrobial Resistance & Infection Control – Springer Journals

Published: Jul 29, 2019

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