|Year : 2020 | Volume
| Issue : 1 | Page : 58-65
Colistin susceptibility testing of gram-negative bacilli: Better performance of vitek2 system than E-test compared to broth microdilution method as the gold standard test
Surojit Das1, Subhanita Roy2, Samadrita Roy2, Gaurav Goelv1, Subir Sinha3, Purva Mathur4, Kamini Walia5, Sanjay Bhattacharya2
1 Department of Bio-Medical Laboratory Science and Management, Vidyasagar University, Midnapore, West Bengal, India
2 Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India
3 Department of Statistics, Tata Medical Center, Kolkata, West Bengal, India
4 Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India
5 Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research, New Delhi, India
|Date of Submission||17-Dec-2019|
|Date of Decision||31-Mar-2020|
|Date of Acceptance||30-Jun-2020|
|Date of Web Publication||25-Jul-2020|
Dr. Sanjay Bhattacharya
Tata Medical Center, 14 MAR (E-W), New Town, Rajarhat, Kolkata - 700 160, West Bengal
Source of Support: None, Conflict of Interest: None
Introduction: Unavailability of optimal susceptibility testing (ST) challenges the clinical use of colistin. Broth microdilution (BMD), which is the reference for colistin ST, is inconvenient for diagnostics. Vitek2 and E-test although technically easier, are no longer recommended. Materials and Methods: For the evaluation of Vitek2 and E-test in reference with BMD, a total of 138 Gram-negative bacilli (GNB) especially carbapenem-resistant isolates from Tata Medical Center, Kolkata, India, were included during 2017–2018. The evaluation was performed only for Enterobacteriaceae (n = 102), but not for non-fermentative GNB (n = 36) due to lack of colistin-resistant (COLR) isolates. Results and Conclusion: Of 138 isolates, meropenem, colistin and dual resistance were detected in 110 (79.7%), 31 (22.5%) and 21 (15.2%) of isolates, respectively. Using the European Committee on Antimicrobial Susceptibility Testing guidelines (susceptible, ≤2 μg/ml), Vitek2 performed better than E-test (essential agreement, 92.2% vs. 63.7%; categorical agreement, 94.1% vs. 93.1%; very major error [VME], 10% vs. 23.3%). However, Vitek2 overcalled resistance than E-test (major error, 4.2% vs. 0%). Considering Chew et al. proposed breakpoints (susceptible, ≤1 μg/ml), VMEs declined for both test (6.7% vs. 10%), but still remained unacceptable. Of eight colistin-heteroresistant isolates, two VME were categorised by Vitek2, one VME was by E-test, and two were uninterpretable. Both Vitek2 and E-test are unreliable. Further studies correlating minimum inhibitory concentrations with clinical outcome are needed to determine the accurate breakpoints for better patient management.
Keywords: Broth microdilution, Clinical and Laboratory Standards Institute, colistin, European Committee on Antimicrobial Susceptibility Testing, Gram-negative bacilli, susceptibility testing
|How to cite this article:|
Das S, Roy S, Roy S, Goelv G, Sinha S, Mathur P, Walia K, Bhattacharya S. Colistin susceptibility testing of gram-negative bacilli: Better performance of vitek2 system than E-test compared to broth microdilution method as the gold standard test. Indian J Med Microbiol 2020;38:58-65
|How to cite this URL:|
Das S, Roy S, Roy S, Goelv G, Sinha S, Mathur P, Walia K, Bhattacharya S. Colistin susceptibility testing of gram-negative bacilli: Better performance of vitek2 system than E-test compared to broth microdilution method as the gold standard test. Indian J Med Microbiol [serial online] 2020 [cited 2020 Aug 14];38:58-65. Available from: http://www.ijmm.org/text.asp?2020/38/1/58/290679
| ~ Introduction|| |
Global rising trend of multidrug-resistant (MDR) organisms, especially carbapenem-resistant (CR) Gram-negative bacilli (GNB), is narrowing the optimal treatment strategies resulting in the revival of a 50-year-old antibiotic such as colistin (polymyxin E) as last-resort for anti-infective therapy., Currently, colistin was reported as a valuable antibiotic with acceptable nephrotoxicity and considerable effectiveness. Of late, sporadic emergence of colistin-resistant (COLR) isolates across the globe including India importantly harbouring mcr-1 gene further threatens the treatment options.,,
Lack of reliable reference susceptibility testing (ST) method of colistin has restricted the evaluation of commercially available tests. In 2016, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial ST (EUCAST) jointly recommended broth microdilution (BMD) as the reference method for minimum inhibitory concentration (MIC) determination of colistin, and abandoned the use of other methods including agar dilution (AD), disk diffusion (DD) and gradient diffusion. Manual BMD is not convenient to use in routine clinical laboratories. The cationic properties of colistin, mediates its adsorption to commonly used labwares albeit at low concentrations, including polystyrene, polypropylene and glass, influences the MICs determined by BMD. The adsorption of colistin to polystyrene wells can be mitigated using cation-adjusted Mueller–Hinton broth (CAMHB) with or without addition of surfactant polysorbate-80 (P-80)., However, the surfactant exhibits a synergistic effect with colistin enhancing its interaction with the bacterial cell membrane without improving the performance of assay. Colistin methanesulphonate, is used in treatment, but cannot be used for ST due to erroneously high MIC values. Finally, the joint CLSI-EUCAST working group has recommended the ISO-20776 standard BMD method for MIC testing of colistin using the sulphate salt of colistin and CAMHB without adding P-80 in non-treated polystyrene trays.,
The development of rapid and reliable colistin ST is a major challenge and urgently needed in diagnostic settings to initiate accurate treatment and reduce the potential toxicities associated with this antibiotic. Several commercial BMD system (Sensititre), gradient diffusion (E-test, MTS) and automated AST systems (Vitek2, MicroScan) were evaluated for use in diagnostics, but none of them met the Food and Drug Administration (FDA) acceptance criteria. Most of them commonly exhibited false susceptible result probably due to the presence of colistin heteroresistant subpopulations., Colistin heteroresistance is defined as colistin-resistant subpopulations which emerge from colistin-susceptible population under colistin pressure and may be evidenced by the presence of skip wells in BMD. Amongst the commercially available MIC-based tests, Vitek2 and E-test were widely accepted for routine use. The Vitek2, is the cost-effective test, provides ST of multiple antibiotics including colistin. E-test provides broader range MICs of colistin, currently is labelled as research-use only. Poor diffusion of large colistin molecule in agar caused errors in interpretation of results and diminished its clinical utility for colistin ST.
Availability of reliable MIC breakpoints is also crucial to confirm any organism as susceptible (S), intermediate (I) or resistant (R) to antibiotic like colistin. Continuous revision of colistin ST breakpoints by various organisations further complicates the interpretation of ST results. Currently, harmonised clinical MIC breakpoints of colistin have been shared by the CLSI and EUCAST for Pseudomonas aeruginosa and Acinetobacter baumannii (S, ≤2 μg/ml)., For Enterobacteriaceae, EUCAST adopts the same breakpoints, while CLSI uses it as epidemiological cut-off values.
In the present study, we evaluated the commonly used Vitek2 and E-test for colistin ST using BMD (without P-80) as reference to judge the accuracy of the methods in clinical decision-making using contemporary GNB isolates during 2017–2018.
| ~ Materials and Methods|| |
A total of 138 clinical GNB especially CR isolates from unique patient for which a colistin MIC test was required by a clinician at Tata Medical Center (TMC) in Kolkata, India, during May 2017 to March 2018 were used in the present study. The isolates comprised Enterobacteriaceae species, namely Escherichia coli (n = 27), Klebsiella pneumoniae (n = 56), Enterobacter cloacae (n = 19) and non-fermentative (NF) GNB species, namely A. baumannii (n = 19) and P. aeruginosa (n = 17). The isolates were recovered from patients with bloodstream (n = 78), urinary tract (n = 23), respiratory tract (n = 21), skin and soft tissue (n = 14) and other (n = 2) infections and identified to species level using Vitek2 (bioMérieux SA, Marcy l'Etoile, France). All isolates were stored frozen at −70°C in brain/heart infusion broth with 15% glycerol for further testing.
Colistin susceptibility testing
Colistin ST was performed by BMD, E-test and Vitek2 system (bioMérieux) for determination of MICs., Quality control was assessed using the strains Escherichia coli ATCC 25922 (colistin MIC, 0.25–1 μg/ml), P. aeruginosa ATCC 27853 (colistin MIC, 0.25–2 μg/ml) and mcr-1 positive E. coli NCTC 13846 (colistin MIC, 4 μg/ml)., BMD testing was performed using untreated 96-well polystyrene microwell-plates (Greiner, Frickenhausen, Germany) containing two-fold dilutions of colistin sulphate salt (concentration range, 0.12–64 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) in CAMHB (BD, Franklin Lakes, NJ, USA). Supplementation with P-80 was not performed, as per CLSI/EUCAST recommendations. Skip well phenomenon was defined as the absence of growth of an isolate at lower antimicrobial concentration(s). A single skip well did not affect the MIC interpretation, while multiple skip wells were considered as uninterpretable according to CLSI guidelines. For skip well phenomenon in BMD panels, the isolates were retested. E-test method using a colistin strip (concentration range, 0.016–256 μg/ml) (bioMérieux) were performed with Mueller–Hinton agar (MHA) (BD) plates in accordance with recommendations of the manufacturers. For E-test, the highest MIC intersection was considered for isolates showing individual colonies within the bioMerieux SA, Marcy l'Etoile, France ZOI. Vitek2 AST N281 susceptibility card (bioMérieux) was used to determine colistin MIC (calling range, ≤0.5 to ≥16 μg/ml) according to the manufacturer's instructions. E-test and Vitek2 MICs between two-fold dilutions were rounded up to the next highest two-fold dilution. Susceptibility to meropenem was also recorded among the isolates using Vitek2.
Polymerase chain reaction detection of mcr-1 gene
The study isolates (n = 138) were screened for the presence of mcr-1 gene by polymerase chain reaction (PCR) using published primer. Boiled DNA template was prepared by dissolving a single colony in 200 μl of distilled water and lysing the bacteria at 95°C for 10 min and collecting the supernatant following centrifugation. PCR amplicons were visualised on 1.5% agarose gel pre-stained with GelRed nucleic acid (Biotium, Inc., Hayward, CA).
Interpretation of results and data analysis
The interpretation of colistin MIC breakpoints (S, ≤2 μg/ml; R, >2 μg/ml) for Enterobacteriaceae, A. baumannii and P. aeruginosa was performed following the EUCAST guideline. In addition, Chew et al. proposed colistin MIC breakpoints (S, ≤1 μg/ml; I, 2 μg/ml, and R, ≥4 μg/ml) for Enterobacteriaceae was validated. Scatter plots were generated with the Vitek2 MIC and E-test MIC results on y axis and BMD MICs on x axis. In reference with BMD, colistin concentration ranging from ≤ 0.5 to ≥16 μg/ml and ≤0.125 to >64 μg/ml was used for Vitek2 and E-test, respectively. Essential agreement (EA; percentage of isolates with MICs within 1 doubling dilution from the reference method MIC; [number of isolates within ± 1 doubling dilution/total number of isolates] × 100) Categorical agreement (CA; percentage of isolates in the same susceptibility category by BMD and the method under evaluation; [number of isolates with same S, I, or R result/total number of isolates] × 100), very major errors (VMEs; false-S result; [number of false-S/total number of R isolates] × 100), major errors (MEs; false-R result; [number of false-R/total number of S isolates] × 100) and minor errors (mEs; related to an I interpretation for either the reference or test method; [number of mEs/total number of isolates] × 100). Performances of the methods were evaluated following the acceptable limits as stated earlier: ≥90% for EA, ≥90% for CA, ≤1.5% for VMEs, ≤3% for MEs and ≤10% for mEs.
Spearman's correlation coefficient (R) was calculated to measure the strength and direction of association between MIC values obtained by Vitek2 and E-test with those of BMD MICs using the computer package R version 3.5.0 for Windows (R programming language, Vienna, Austria). Chi-square test with Yates correction and Student's t-test were used to determine the significance of difference. Differences were considered statistically significant at a P < 0.05.
| ~ Results|| |
Colistin susceptibility testing
Colistin ST was performed among Enterobacteriaceae (n = 102) and NF GNB (n = 36) isolates. Of the 138 isolates, 110 (79.7%), 31 (22.5%) and 21 (15.2%) were MEMR, COLR and dual COLR-MEMR, respectively [Figure 1]a. Noticeably, 90.5% (19/21) of the dual COLR-MEMR isolates were K. pneumoniae. On the contrary, all COLRE. cloacae isolates (n = 6) were susceptible to meropenem. Although overall association between meropenem and colistin resistance was not statistically significant (χ2 = 2.65; P = 0.103), but it was found statistically significant in K. pneumoniae (χ2 = 40.67; P < 0.0001). Higher isolation of COLR isolates from non-blood specimens (19/60, 31.7%) than blood (12/78, 15.4%) was found to be statistically significant (χ2 = 4.27; P = 0.039) [Figure 1]b. The MIC distribution of colistin, stratified by bacterial species, is presented in [Table 1]. Colistin was found to be effective (BMD MIC90, ≤2 μg/ml) against E. coli, A. baumannii and P. aeruginosa isolates, while minimal activity (BMD MIC90, ≥32 μg/ml) was noticed in both K. pneumoniae and E. cloacae [Table 1].
|Figure 1: Colistin susceptibility among Gram-negative bacilli isolates in (a) association with meropenem resistance and (b) blood and non-blood specimens. Colistin and meropenem susceptibility were determined by broth microdilution and Vitek 2, respectively. COL-S: Colistin-susceptible, COL-R: Colistin-resistant, MEM-R: Meropenem-resistant, B: Blood; NB: Non-blood|
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|Table 1: Distribution of colistin minimum inhibitory concentration by broth microdilution, Vitek 2 and E-test for the tested isolates|
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Presence of mcr-1 gene
None of the isolates (n = 138) were found to be positive for mcr-1 gene.
Evaluation of colistin minimum inhibitory concentration susceptibility test results
Among the Enterobacteriaceae (n = 102), the MIC values obtained by Vitek2 and E-test were evaluated in reference with BMD method using the EUCAST breakpoints [Table 2] and [Figure 2]a. Vitek2 showed acceptable EA rate (>92.2%) and a very strong positive correlation (R = 0.895) with the MIC results obtained by BMD (t- test, 20.08; P < 0.0001) [Table 2]. Although a strong positive correlation (R = 0.678) existed between E-test and BMD (t- test, 9.23; P < 0.0001), E-test had lower EA rate (63.7%). Both the test yielded acceptable limit of CA rate (>90%) [Figure 2]a. In terms of error, both Vitek2 (10%) and E-test (23.3%) exhibited VME much higher than acceptable limit [Figure 2]a. E-test had no ME, but Vitek2 showed marginally higher ME (4.2%).
|Table 2: Performance characteristics of colistin susceptibility testing methods in comparison to broth microdilution|
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|Figure 2: Scatter plots of colistin susceptibility testing. Comparative evaluations of Vitek 2 and E-test with broth microdilution using (a) European Committee on Antimicrobial Susceptibility Testing 2017 and (b) Chew et al., 2017 proposed breakpoints. Solid lines indicate the minimum inhibitory concentration breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing in 2017, and dashed line represent proposed minimum inhibitory concentration breakpoint in the present or earlier study. CA, very major error, major error and minor error have been mentioned below each scatter plot. Susceptible, intermediate and resistant isolates represented by light grey, dark grey and black capped line, respectively|
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When considering Chew et al. proposed breakpoints, the CA rate reduced still in acceptable limit, but the error rates improved [Figure 2]b. The VME rates of Vitek2 (6.7%) and E-test (10%) reduced, but still existed in unacceptable limit [Figure 2]b. Vitek2 showed acceptable rates of ME (3%) and mE (4.9%). E-test had acceptable mE (7.8%) without any ME.
Among the NF GNB (n = 36), the EA rate was found just below acceptable limit for both Vitek2 and E-test [Table 2]. Only one isolate (A. baumannii) was COLR [Table 1]. Error rates were not possible to calculate due to the lack of sufficient resistant isolates.
Presence of skip well(s) in broth-microdilution or isolated colonies in agar diffusion test
A total of eight isolates showed single or multiple skip-well(s) by BMD and/or growth of individual colonies up to the edge of the colistin E-test within a ZOI [Table 3]. The isolates showing skip wells were confirmed by repeated testing. Six of them were E. cloacae isolates. Single skip well was found in four isolates by BMD, while the growth of individual colonies was detected in six isolates by agar diffusion. Of note, VME by Vitek2 and E-test showed two and one VME among the colistin-heteroresistant isolates, respectively. Two isolates (one E. cloacae and one P. aeruginosa) had to be rejected because they exhibited uninterpretable MIC results due to the presence of multiple skip-wells in BMD.
|Table 3: List of isolates showing growth of individual colonies within E-test zone of inhibition or skip-well(s) phenomenon|
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| ~ Discussion|| |
Limited therapeutic options have forced clinicians and microbiologists to reappraise the clinical application of colistin for treating infections caused by MDR, especially CR GNB. To circumvent the crisis, both VME (false-S) and ME (false-R) of colistin ST should be equally weighed. Clinical laboratories need to produce correct colistin susceptibility results having good concordance with reference method for clinicians. In this study, we evaluated colistin MICs obtained by Vitek2 and E-test among contemporary Enterobacteriaceae and NF GNB isolates at our institution during 2017–2018.
Earlier studies documented about 2% colistin resistance among GNB, but high rate (>20%) was found in CR isolates including India, especially in K. pneumoniae.,,,, Higher isolation rate (22.5%) of COLR study isolates might be associated with the prevalence of meropenem resistance (79.7%) among the isolates, but the association was not statistically significant [Figure 2]. Majority (90.5%) of the dual COLR-MEMR study isolates were K. pneumoniae. Bacteraemia due to COLR GNB especially K. pneumoniae was documented in earlier studies and associated with high mortality., Amongst the 12 COLR blood isolates, K. pneumoniae was isolated in 8 (66.7%) cases in the present study [Figure 1]b. Colistin was found to be more effective against E. coli, A. baumannii and P. aeruginosa isolates than K. pneumoniae and E. cloacae as reported earlier.,
The evaluation of commercially available colistin ST methods revealed contradictory results importantly for VME rate due to variation in study design especially reference methodology and colistin susceptibility profile of target isolates., Following current recommendations, we evaluated Vitek2 and E-test using sizeable number of Enterobacteriaceae isolates (n = 15, 14.7%) having MICs near the breakpoints (MIC, 1 μg/ml to 8 μg/ml). For NF GNB, the evaluation of the methods was not performed due to the lack of resistant isolates. Retrospective studies might misguide about the interpretation of discrepancies rates of different colistin ST due to loss of resistance or change in MICs during subculture and storage. Hence, we designed the study in prospective mode to minimise the deviations of MICs.
The Vitek2 was evaluated in limited studies for Enterobacteriaceae showing high rate of EA and CA in acceptable limit without any VME,, but few researchers strictly advised not to use Vitek2 due to VME as high as 36%., The system also had tended to overcall resistance (ME) as reported earlier. In spite of high concordance with BMD (R, 0.895) and acceptable EA rates (92.2%) in this study, the Vitek2 was found to be unreliable due to unacceptable rate VME (10%) and ME (4.2%). On the other hand, several researchers warned the clinical use of E-test due to its low rates of agreements (<50%) and high rates of errors (VME, >40%; ME, >10%).,,,, The present study also showed EA of 63.3% and VME of 23.3% reflecting the unreliability of E-test among Enterobacteriaceae. In contrast, Maalej et al. reported good concordance of E-test using AD as reference amongst Enterobacteriaceae without any VME. Poor concordance (R, 0.678) was observed between BMD and E-test MICs among Enterobacteriaceae study isolates as reported earlier.E-test was prone to underestimate the level of colistin resistance depending on the manufacturer of MHA. The present study also highlighted the issue of E-test MICs by ≤2-fold decrease among more than 36% of isolates [Table 2]. This might explain the reason of poor concordance.
The primary purpose of clinical breakpoints is to predict clinical outcomes associated with the disease, but not to detect the underlying resistance mechanisms. However, the sporadic emergence of plasmid-mediated mcr-1 gene especially in E. coli and K. pneumoniae is associated with either lower level of colistin resistance (MIC, 4–8 μg/ml) or even susceptible (MIC, ≤1 μg/ml) phenotype and may contribute to treatment failure.,, Hence, Chew et al. used lower breakpoint at ≤1 μg/ml for evaluation and reported good correlation with the presence of mcr-1 gene which in turn improved the discrepancies of various ST. Using the Chew et al. proposed breakpoints, the discrepancy rates especially VMEs were also found to decline for both Vitek2 and E-test, but still did not satisfy the FDA criteria. It would be interesting to explore the association between colistin MICs and mcr-1 gene, but none of the study isolates were positive for mcr-1.
Colistin heteroresistance was mainly documented in A. baumannii and E. cloacae than in P. aeruginosa, and K. pneumoniae further complicating the interpretation of ST results.,,, The heteroresistant isolates showed growth of individual colonies with ZOI of E-test and skip well phenomenon in BMD., In the present study, similar phenomenon was noticed only in eight isolates predominantly in E. cloacae, but none of the K. pneumoniae had the heteroresistant profile [Table 3]. COLR including heteroresistant isolates were reported to be reliably detected by Iso-Sensitest agar instead of MHA. Recently, Nordmann et al. reported 100% sensitivity and specificity for the detection of polymyxin-resistant GNB using a novel universal medium. As we used MHA, this might be the reason for getting lower number of COLR isolates (n = 24; 17.4%) by E-test. Vitek2 reported to produce false susceptible results in hetero-resistant populations. Although limited number of study isolates belonged to heteroresistance profile, Vitek2 also showed false susceptible results among them. No interpretation was available for isolates showing multiple skip wells, hence, two study isolates were not included for evaluation.
| ~ Conclusion|| |
The present study highlights the importance of optimal colistin ST amidst CR isolates. Vitek2, performs better than E-test, but remains unreliable due to high VME. Chew et al. proposed stringent MIC breakpoints may be useful in clinical settings, but requires rigorous validation especially using mcr-1 positive and heteroresistant isolates. Further studies correlating MICs with clinical outcome are needed to determine the accurate breakpoint to lead patient management.
We would like to thank A. D. Misra, B. Jana, S. Pal and other technical staffs of TMC, Kolkata for providing assistance during the study.
Financial support and sponsorship
This study was supported by the Indian Council of Medical Research, New Delhi, India [grant number AMR/RC/63/2014-ECD-II].
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]