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INTRODUCTION
The rapid spread of carbapenemase-producing Enterobacterales (CPE) worldwide is a serious threat to public health [1], as it is capable of spreading resistance genes through horizontal transfer and has been related to several outbreaks [2]. In Korea, reporting CPE infections to the Korean Center for Disease control and Prevention (KCDC) has been made mandatory for hospitals since 2012 (http://www.kdca.go.kr/). According to the KCDC, there were 11,954 carbapenem-resistant Enterobacteriaceae (CRE) cases in 2018 and 15,369 cases in 2019. Among the 2019 cases, 57.8% (8,887/15,369) were confirmed as CPE infections [3]. For infection control, rapid identification of patients colonized with CPE has become a routine clinical aim in many parts of the world and is recommended by public health organizations [4]. Various methods to detect CPE in rectal swabs, including culture with specific chromogenic media and commercial molecular tests, have been used [5]. Although concomitant culture is necessary for bacterial species identification and epidemiological typing, it requires more than two days of turnaround time. To overcome this limitation, molecular tests have been developed with reduced processing time to rapidly implement proper infection control measures [6, 7]. As a rapid molecular test, the Xpert Carba-R assay is capable of detecting five carbapenemase genes (blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP-1) in approximately one hour and has been used in our laboratory since July 2017 for active surveillance of CPE. In this study, we evaluated the performance of the Xpert Carba-R assay compared with the conventional method culture plus in-house PCR.
MATERIALS AND METHODS
1. Study design
We retrospectively analyzed the routine test results of the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA) and the conventional method culture plus in-house PCR from August 2018 to July 2020. Screening for CPE was performed using rectal swab specimens from patients with high risk of intestinal colonization with CPE in accordance with the infection control policy of Seoul St. Mary’s Hospital. Screening was performed for all patients admitted to the intensive care unit or the general ward if the patient had been transferred from another hospital after a stay longer than 1 week within the last 4 weeks. For all patients admitted to the intensive care unit, testing was performed at admission, after every subsequent seven days, and before discharge.
The rectal swab specimens were collected in eSwab 480CE (Copan Diagnostics, Inc., Brescia, Italy) and submitted to a clinical microbiology laboratory. The Xpert Carba-R assay and culture plus in-house PCR were performed simultaneously, as described below.
This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, Seoul, South Korea (KC21RASI0674).
2. Xpert Carba-R assay
The rectal swabs were processed using Xpert Carba-R assay, a real-time PCR assay designed to detect the five most common carbapenemases, i.e., blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP-1. The assay was performed according to the manufacturer's instructions. The transport media was sent to the laboratory and vortexed, and a 100 μL sample was mixed with Xpert Carba-R assay sample buffer. After vortexing the mixture, 1.7 mL of the sample buffer was transferred to an Xpert Carba-R assay cartridge and was placed on the GeneXpert™ system.
3. Reference method: Confirmation of culture positivity using in-house PCR
For conventional culture method, 100 μL of each specimen was placed in 8 mL of MacConkey broth (BD, Franklin Lakes, NJ, USA) containing a 10 μg ertapenem disk (BD). After 24 hours and 48 hours of incubation at 37°C, if the solution turned yellow or turbid, an aliquot of 100 μL was removed and inoculated onto MacConkey agar (Asan Pharmaceutical, Seoul, Korea) and CHROMagar mSuperCARBA agar (CHROMagar, Paris, France) plates. Next, 10 μg ertapenem disks were placed in the first and second quadrants of MacConkey agar for detection of carbapenem-resistant isolates. The colonies that grew within 27 mm of the ertapenem disks after overnight incubation at 37°C were suspected as CRE [8].
Additionally, for cases showing positive results in the Xpert Carba-R assay, 100 μL of each specimen was inoculated directly onto a CHROMagar plate for more rapid identification of CPE species. The species identification was performed using matrix assisted laser desorption ionization time-of-flight (MicroIDSys, ASTA, Suwon, Korea), and the antimicrobial susceptibility test was performed using Vitek-2 (bioMérieux, Marcy L’Etoile, France). To confirm the presence of carbapenemase genes, colonies were suspended in 1 mL distilled water in a 1.5 mL micro tube and vortexed. After washing, the pellet was boiled for 20 minutes for DNA extraction, followed by PCR using a C1000 Thermal Cycler Chassis (Bio-Rad, Hercules, CA, USA) to detect the most common five genes, blaSIM, and blaGES. The primer sequences used for PCR are described in previous studies [9, 10].
4. Data analysis
Growth of CPE either on MacConkey agar or CHROMagar with carbapenemase gene confirmation using in-house PCR was defined as true-positive. No growth of CPE was defined as true-negative. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) including 95% confidential interval (CI) were calculated for all test results [11]. Statistical analysis was performed using the MedCalc Statistical Software version 18 (MedCalc Software Bvba, Ostend, Belgium; http://www.medcalc.org; 2018). Differences in the threshold cycle (Ct) values between CPE and non-CPE (false-positive results of Xpert Carba-R assay) specimens were analyzed using the Mann-Whitney U-test. P values <0.05 were considered significant. Samples that found positive result with the Xpert Carba-R assay, 24 hours earlier-inoculation was done on CHROMagar directly. And the samples were compared with that inoculated on MacConkey broth. For analysis of prevalence, only one specimen per patient was used. The Chi-square test was used to compare culture results of specimens found to be true- or false-positives in the Xpert Carba-R assay. This test was also used to compare the results of the prevalence of CPE during the two periods.
RESULTS
1. Xpert Carba-R assay results
A total of 26,178 specimens from 12,889 patients were tested from August 2018 to July 2020.
In the Xpert Carba-R assay, 6.2% positive cases (1,615/26,178 specimens) were detected. Furthermore, in the reference method, 5.8% positive cases (1,525/26,178 specimens) were detected (Table 1).
2. Comparison of the Xpert Carba-R assay with the reference method
Table 2 shows the overall performance of the Xpert Carba-R assay compared to the reference method. Sensitivity, specificity, PPV, and NPV of the Xpert Carba-R assay were 97.4% (95% CI, 96.5% to 98.1%), 99.4% (95% CI, 99.3% to 99.5%), 91.8% (95% CI, 90.5% to 93.0%), and 99.8% (95% CI, 99.7% to 99.8%), respectively. There were 26,178 specimens analyzed for five individual genes, providing 130,890 test results. The results were defined as positive for the Xpert Carba-R assay if at least one carbapenemase gene was detected.
3. Discrepant results
Table 3 shows discrepant results between the Xpert Carba-R assay and the reference method. Here, 39 cases that showed negative in the Xpert Carba-R assay but showed positive in the reference method were considered false-negative results of the Xpert Carba-R assay. Most of these were blaKPC-producing Klebsiella pneumoniae (N=29). In contrast, 131 specimens yielded false-positive results in the Xpert Carba-R assay. Among these, blaIMP-1 was the most common gene (N=48), followed by blaNDM, blaKPC, and blaOXA-48. In 90 of the 131 cases, CRE did not grow. In the remaining 41 cases, the carbapenemase genes were detected in non-fermentative bacilli, most of which were identified as Pseudomonas aeruginosa (23/41, 56.1%), followed by Acinetobacter baumannii (10/41, 24.4%).
Compared to the true-positive cases detected in the Xpert Carba-R assay, the median Ct value of false-positive cases was significantly higher (24.3 vs. 35.0, P<0.001, Mann-Whitney U-test) (Fig. 1). According to carbapenemase type, the median Ct values of true-positive groups were 25.4, 24.9, 23.3, 26.0, and 32.3 for blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP-1, respectively. The median Ct values of false-positive groups were 29.0, 32.0, 34.1, 34.2, and 35.0 for blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP-1, respectively. Differences in Ct values were statistically significant for all five genes, blaKPC (P=0.003), blaNDM (P<0.001), blaOXA-48 (P<0.001), blaVIM, (P=0.002), and blaIMP-1 (P=0.033).
Additionally, in the MacConkey broth culture, one blaGES-producing K. pneumoniae and two blaSIM-producing C. freundii were found using the in-house PCR method.
4. Prevalence of CPE
The prevalence of CPE and CRE were 1.7% (220/12,889) and 5.4% (695/12,889), respectively. Klebsiella pneumoniae (108/220, 49.1%) was the most common species, followed by E. coli (68/220, 30.9%) and C. freundii (17/220, 7.7%). blaKPC was the most common carbapenemase gene (123/220, 55.9%), followed by blaNDM (56/220, 25.4%), blaOXA-48 (27/220, 12.2%), and blaNDM/OXA-48 (9/220, 4.1%).
The prevalence of CPE from August 2018 to July 2020 is shown on Fig. 2. During the first period (2018.08-2019.07) of the study, the prevalence of CPE was 1.3% (87/6,546), and the proportion of CPE isolates among CRE isolates was 38.1% (87/228). Of these 87 isolates, 41 harbored the blaKPC gene, while the remaining isolates carried blaNDM (N=22) or blaOXA-48 (N=14) genes. Escherichia coli (37/87, 42.5%) was the most common species, followed by K. pneumoniae (25/87, 28.7%) and C. freundii (8/87, 9.2%). During the second period (2019.08-2020.07) of the study, the prevalence of CPE increased to 1.7% (133/6,343), and the proportion of CPE patients among CRE patients was 28.4% (133/467) due to the increase in the number of CRE patients. Of these 133 CPE cases, 82 harbored blaKPC, followed by the remaining cases carrying blaNDM (N=34) or blaOXA-48 (N=13). Klebsiella pneumoniae (83/133, 62.4%) isolates were the most common species during this period, followed by E. coli (31/133, 23.3%) and C. freundii (9/133, 6.8%). The most common species over the two years of the study showed different and increased blaKPC-producing K. pneumoniae up to 53.3% (71/133) from 20.6% (18/87). The results of the Chi-square test for carbapenemase gene and species showed P-value<0.05 for blaKPC-producing K. pneumonia; no other combination showed significant result.
DISCUSSION
The Xpert Carba-R assay detected 1,615 positive targets (827 blaKPC, 461 blaNDM, 371 blaOXA-48, 20 blaVIM, and 68 blaIMP-1) and multiple genes in 126 specimens (Table 1). Among multiple genes detected, blaNDM+blaOXA-48 were most common (73.8%, 93/126), consistent with previous studies in Korea [3, 12], and were followed by blaKPC+blaIMP-1 (6.3%, 8/126) and blaKPC+blaVIM (4.8%, 6/126). Species of blaNDM,+blaOXA-48 were E. coli, except for three specimens (two specimens for E. coli and Klebsiella oxytoca, and one specimen for E. coli and Kluyvera cryocrescens). In other multiple gene detected carbapenemases (33/126), blaKPC was the most common gene (25/33).
The Xpert Carba-R assay demonstrated high sensitivity and specificity (97.4% and 99.4%, respectively). Our results are in agreement with results from several studies showing excellent performance for each carbapenemase [13, 14]. The PPV for blaKPC was 97.4% in our study; however, Tato et al. [13] reported relatively low PPV for blaKPC (87.9%). Higher PPV in our study for blaKPC might be due to higher sensitivity of our reference method [15-17] or higher prevalence of blaKPC in the studied population. Higher sensitivity of CHROMagar than MacConkey agar with a meropenem disk for CPE screening was reported previously [5], and inclusion of CHROMagar as reference method might have increased the specificity and PPV of the Xpert Carba-R assay. This finding indicates that the high sensitivity of the reference method is critical for evaluation of molecular methods and that studies of the performance of the Xpert Carba-R assay can show differences in results [13, 18]. Additionally, 89 out of 1,484 CPE specimens showed negative results with the MacConkey broth after 24 hours incubation, while seven showed negative results with MacConkey broth after 48 hours incubation. Seven specimens were only confirmed using CHROMagar, four blaKPC-producing E. coli, one blaOXA-48-producing E. coli, and two blaKPC-producing K. pneumoniae. In contrast, no isolate was identified using MacConkey broth only. Therefore, inoculation of rectal swabs onto a CHROMagar plate allows faster and more accurate identification of CPE, which could prevent dissemination.
The PPV for blaIMP was 29.4%, which was lower than the PPV values of other carbapenemase genes, partly because of the low prevalence of blaIMP in Korea [3]. Additionally, blaIMP was mainly detected in non-fermentative bacilli, consistent with a previous study [19].
Among the 39 false-negative specimens, 29 harbored blaKPC-producing K. pneumoniae (Table 3). The small number of organisms in the rectal swab specimens might be the cause of false-negatives in these specimens. This is supported by the findings that most (94.0%, 1,395/1,484) of the true-positive specimens showed growth after 24 hours incubation in MacConkey broth, whereas as many as 30.8% (12/39) of the false-negative specimens showed growth after 48 hours incubation (Chi-square test, P-value<0.001).
Among the 131 false-positive results, growth of carbapenem-resistant isolates was not observed in 90 cases (68.7%). Among the remaining 41 cases, growth of P. aeruginosa and A. baumannii was observed in 23 and 10 cases, respectively (Table 3). A high median Ct value of 35.0 was found for the false-positive results (Fig. 1). This could be explained by a low level of target gene expression of the organism or the possibility of medicinal treatment or modified sequence of the target gene. Compared to the true-positive cases detected using the Xpert Carba-R assay, the median Ct value of false-positive cases was significantly higher (24.3 vs. 35.0, P<0.001) (Fig. 1).
In our study, rare carbapenemase genes (blaSIM and blaGES) were detected from three isolates; the blaSIM was found in two C. freundii isolates, while the blaGES was found in one K. pneumoniae isolate. The prevalence of blaGES and blaSIM genes was very low in our study (0.2%), in line with the population results of studies conducted in Korea, the United States, and Europe [12, 20]. However, it is essential to verify the absence of blaGES and blaSIM genes to prevent dissemination through horizontal spread. A recent study in India about dissemination of plasmids encoding carbapenemases in gram-negative bacteria showed blaGES as the third most common gene (11 of 37, 29.7%, 6 for blaGES-1 and 5 for blaGES-9) in E. coli.
One of the limitations of our study is that we did not perform a direct PCR or sequencing of the MacConkey broth. Tato et al. [13] demonstrated that 11 of 18 specimens previously classified as false-positives using the Xpert Carba-R assay were reclassified as true-positives after discrepant sequencing analysis of the MacConkey broth. This might be due to low bacterial load of carbapenemase in the rectal swab specimens or failure of carbapenemase-harboring species to recover using the initial culture method. And Our study did not include the new emerging blaOXA variants, such as blaOXA-232 [21].
However, to the best of our knowledge, this study included the largest number of clinical specimens. Based on the results obtained, we confirmed high NPV (99.8%) of the Xpert Carba-R assay, with the prevalence of up to 27%. Considering the high sensitivity (97.4%) and NPV (99.8%) of the Xpert Carba-R assay, for cases showing culture positivity, confirmatory PCR might be omitted in regions with a very low prevalence of other carbapenemases not included in the Xpert Carba-R assay such as blaGES, and only species identification and antimicrobial susceptibility test would be needed for identification of CRE.
In conclusion, the prevalence of both CPE and CRE was higher in 2020 compared to that in 2019. The Xpert Carba-R assay showed excellent performance and can be used as a useful tool for active surveillance for CPE.
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