|Year : 2017 | Volume
| Issue : 4 | Page : 365-369
A comparative study of modified Hodge test and Carba NP test for detecting carbapenemase production in Gram-negative bacteria
Sayoni Datta1, Rupali Dey2, Jayanta Bikash Dey1, Srijita Ghosh1
1 Department of Microbiology, Bankura Sammilani Medical College, Bankura, West Bengal, India
2 Department of Microbiology, Burdwan Medical College and Hospital, Burdwan, West Bengal, India
|Date of Submission||14-Sep-2016|
|Date of Acceptance||10-Jan-2017|
|Date of Web Publication||4-Sep-2017|
Mohini Apartment, Block-A, Flat-B-2, H/I-36, S. L. Sarani, Baguiati, Kolkata - 700 059, West Bengal
Source of Support: None, Conflict of Interest: None
Objective: The aim of this study was to evaluate the efficacy of the modified Hodge test (MHT) and Carba NP test for the detection of carbapenemase production on Gram-negative bacilli (GNB). Materials and Methods: A total of 816 well-characterized GNB isolated from various clinical samples received from both out- and hospitalized patients were included in the study. Out of those, 16.18% (132/816) were carbapenem-resistant on screening test. MHT and Carba NP test were carried out on all these carbapenem-resistant GNB isolates. Results: Out of the 132 isolates screened resistant during the study period, 75% (99/132) were positive for MHT and 86.4% (114/132) showed the presence of carbapenemase by Carba NP test. Conclusion: The advantage of the Carba NP test over MHT is that it is a rapid test where the results can be read in short time and it is easy to perform. The number of detected carbapenemase producers was significantly higher by Carba NP test though this finding can only be validated by molecular analysis of the strains.
Keywords: Antibiotic resistance, Carba NP test, carbapenemase, modified Hodge test, phenotypic detection
|How to cite this article:|
Datta S, Dey R, Dey JB, Ghosh S. A comparative study of modified Hodge test and Carba NP test for detecting carbapenemase production in Gram-negative bacteria. Med J DY Patil Univ 2017;10:365-9
|How to cite this URL:|
Datta S, Dey R, Dey JB, Ghosh S. A comparative study of modified Hodge test and Carba NP test for detecting carbapenemase production in Gram-negative bacteria. Med J DY Patil Univ [serial online] 2017 [cited 2023 Mar 24];10:365-9. Available from: https://www.mjdrdypu.org/text.asp?2017/10/4/365/213930
| Introduction|| |
Carbapenemases are β-lactamases that hydrolyze penicillins, in most cases cephalosporins, and to varying degrees carbapenems and monobactams.
Carbapenem group of drugs is very effective in treating bacterial infections, can often save lives in case of multidrug resistant infection, especially healthcare-associated infections. For this reason, resistance to carbapenems poses a major challenge to the health-care system, by limiting the options of antibiotics available to treat these infections. Carbapenemase production in Gram-negative bacilli (GNB) is being reported globally at increasing rates. A variety of carbapenemases has been reported in Enterobacteriaceae such as KPC (Ambler Class A), metallo-β-lactamases (MBL) of VIM-, IMP- and NDM (New Delhi MBL)-type (Ambler Class B), and OXA-48-types (Ambler Class D). Detection of Gram-negative bacteria (GNB) that produce carbepenemase has epidemiological significance in controlling further transmission. Laboratory strategies for carbapenemase detection in routine AST consist of a screening test and confirmatory test. Clinical and Laboratory Standards Institute (CLSI) recommended Modified Hodge test (MHT) and European Committee on Antimicrobial Susceptibility Testing recommends Carba NP test as the confirmatory test. Carba NP test has been added in CLSI 2015.
The MHT is a phenotypic method for detection of carbapenemase production. This test lacks specificity and there is delay in obtaining the results (24–48 h) after isolation of a bacterial colony. It also has drawbacks such as false detection of carbapenemase production  and poor sensitivity for NDM-1 detection.
A rapid chromogenic carbapenemase detection assay, the Carba NP test, based on hydrolysis of the β-lactam ring of imipenem, was described by Patrice Nordmann, Laurent Poirel, and Laurent Dortet, which was first published in 2012. The principle of the test was that in vitro hydrolysis of a carbapenem leads to change of pH (decreased pH) that brings about visible color change of the medium from red to yellow/light orange with phenol red indicator. The carbapenemase producers which the Carba NP test fails to detect, have been found by molecular analysis to be mainly of OXA-48-like producing strains, whereas MHT is less reliable to detect other types of carbapenemases as well.
There is not enough data showing the prevalence of carbapenemases and efficacy of various methods for their detection in Eastern India. The study was done from July 2014 to December 2015. We selected MHT and Carba NP test to detect the carbapenemases in GNB isolates. This study was designed to evaluate the efficacy of the MHT and Carba NP test for the detection of carbapenemase production on clinically isolated GNB.
| Materials and Methods|| |
After ethical clearance from the Institutional Ethical Committee, clinical samples received at bacteriology laboratory including urine, pus, wound swab, blood, sputum, and various body fluids from patients admitted in wards, Intensive Care Units, and patients attending various OPD were included in the study.
The samples were inoculated in suitable culture media-MacConkey agar (Himedia, Mumbai, India), blood agar, nutrient agar (Himedia, Mumbai, India). After overnight incubation at 37°C, culture media were examined for bacterial growth. Gram stain was done from bacterial colonies and motility test for GNB was carried out by hanging drop preparation. The identification of isolates was done following standard identification procedure for GNB.
Antibiotic susceptibility test of each GNB was carried out following the CLSI guidelines, on Mueller–Hinton agar (MHA) using antibiotic disk containing meropenem 10 μg and imipenem 10 μg (by Kirby Bauer's disk diffusion technique). After overnight incubation at 37°C, on the next day, GNB isolates, which showed inhibition zone diameter of ≤19 mm to meropenem or imipenem, were selected for the study.
Among a total of 816 well-characterized nonrepetitive GNB isolates, 132 strains which showed resistance to carbapenems were used for this study, out of which 118 were Enterobacteriaceae and 14 were nonfermenters. Phenotypic detection for the production of carbapenemases was carried out on each of these isolates by the MHT on MHA plate following CLSI guidelines. After overnight incubation at 37°C, MHA plates were examined for enhanced growth around the test or control organism streak at the intersection of the streak and the zone of inhibition [Figure 1]. Enhanced growth was considered as positive for carbapenemase production. No enhanced growth was considered negative for carbapenemase production. MHT was performed twice on each isolate.
All the above-selected strains were subjected to growth in MHA using carbapenem disk as per the guidelines  for standardizing strains before performing Carba NP test. Carba NP test was done with all the strains, according to guidelines published by Nordmann et al. and also with few modifications described herewith. In the Carba NP test, as in the original method proposed in the first publication  by Nordmann et al., one calibrated loop (10 μL) of test strain was taken directly from antibiotic susceptibility plate and resuspended in 100 μL Tris-Hydrochloride (HCl) (Himedia, Mumbai, India) 20 mol/L lysis buffer. The suspension was vortexed for 1 min, incubated at room temperature for 30 min, and then centrifuged at 10,000 ×g at room temperature for 5 min. Phenol red solution 0.5% (wt/vol) was prepared, 2 mL of this solution was mixed with 16.6 mL of distilled water, and final pH was adjusted to 7.8 by adding drops of 1N sodium hydroxide. A solution was prepared, 1 ml of which contained 3 mg of imipenem monohydrate, pH 7.8, phenol red solution, and 0.1 mmol/L ZnSO4. After this, 100 μL of this solution was added to the wells of a microtiter tray, and 30 μL of the supernatant (containing bacterial extract) from each strain was mixed in separate wells. The microtiter tray was incubated at 37°C for a maximum of 2 h and then observed for change of color of the solution. Carbapenemase-producing strains caused change of color from red to yellow or light orange.
Several modifications of Carba NP test were published since the first one,,, which included using whole bacterial cells rather than supernatant after lysis, use of eppendorf tubes rather than microtiter tray, using increased concentration of imipenem and using imipenem-cilastatin combination which has more use in clinical practice. In a later work published by Dortet et al., a modification of Carba NP test-the Carba NP II test was done using various solutions of phenol red, ZnSO4, imipenem monohydrate, tazobactum, and ethylene diamine tetraacetic acid. Ambler class of carbapenemases could be detected in this Carba NP II test. After this, in another modification of the Carba NP test by Dortet et al., 1.5 ml eppendorf tubes instead of 96-well microtiter plate, and reduced amount of bacteria was used. According to the author, these modifications eliminated the need for centrifugation step. In a recent work done by Pasteran et al., he designed a novel protocol (CNPt-direct), which was easier to perform using bacterial colonies instead of bacterial extracts, which was more sensitive than Carba NP test mentioned in CLSI, which was promising as most of the OXA-type producers were tested positive, owing to better detection of this enzyme. Here, the sources of carbapenems used were 6 mg/ml imipenem or 12 mg/ml imipenem-cilastatin in injectable form.
The original protocol published by Nordmann et al. and the modification of the Carba NP test, i.e., using eppendorf tubes as recommended by Dortet et al., were performed on the GNB isolates. While using eppendorf tubes, the volume of the final solution in 1.5 ml eppendorf tube was less to interpret visibly, so the amount was increased for each of the supernatant, Tris HCL, and solution A (described later) to three times in the final reaction. Solution A was prepared as follows: Phenol red solution 0.5% (wt/vol) was prepared, 2 mL of this solution was mixed with 16.6 mL of distilled water, final pH was adjusted to 7.8 by adding drops of 1N sodium hydroxide. To this solution, 180 μL of zinc sulfate solution (10 mmol) was added. Solution A was mixed with imipenem powder (6 mg/mL-reconstituted freshly in each day of test). From the lysis buffer solution, 90 μL of the supernatant (containing bacterial extract) from each strain was mixed in two eppendorf tubes with (i) 300 μL of-solution A + imipenem-in one tube and (ii) 300 μL of solution A in another tube (control). Carba NP test was read after incubation at 37°C for 2 h. Each tube was examined for any change of color. Carbapenemase production was noted as change of color from red to yellow or light orange. No carbapenemase production was noted when the solution remained red [Figure 2]. The Carba NP test was also performed with 12 mg/ml imipenem-cilastatin solution. The original protocol  and each of the two modifications were performed thrice. The number of screened strains which were MHT positive and those which showed the presence of carbapenemase activity by Carba NP test were recorded for comparison.
Statistical analyses of the data were done using SPSS Statistics 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. IBM Corp., Armonk, NY, USA). Qualitative and quantitative data were expressed as frequency. Association between two or more qualitative variables was analyzed using Chi-square test. A two-sided P < 0.05 was considered to be statistically significant. Measure of agreement (kappa) value was also calculated.
| Results|| |
A total of 816 well-characterized GNB were isolated during the study period, out of which 16.18% (132/816) isolates showed resistance to carbapenems on screening test. Among the carbapenem resistant GNB isolates, 62.12% (82/132) were Klebsiella pneumoniae, 7.58% (10/132) were Klebsiella oxytoca, 16.67% (22/132) were Escherichia coli, 3.03% (4/132) were Citrobacter freundii, and 10.61% (14/132) were Pseudomonas aeruginosa.
Out of the 132 GNB isolates screened to be resistant, 75% (99/132) were positive for MHT and 86.4% (114/132) showed the presence of carbapenemase activity by Carba NP test [Table 1].
|Table 1: Gram-negative bacilli isolates showing results for modified Hodge test and Carba NP test|
Click here to view
Among the 132 GNB isolates, 71.97% (95/132) were positive for both MHT and Carba NP test, 10.6% (14/132) showed negative results to both the tests, 14.39% (19/132) were MHT negative, but Carba NP test positive and 3.03%(4/132) were MHT positive but Carba NP test negative. The result was statistically significant (P < 0.001). Measure of agreement (kappa) value was less than 0.001.
Carbepemase detection by Carba NP test and MHT for individual GNB was also measured [Table 1], which showed maximum increase in percentage of detection in P. aeruginosa, i.e., 28.57% (4/14), followed by K. oxytoca (20% [2/10]), E. coli (18.18% [4/22]) and K. pneumoniae [6.1% (5/82)]. For C. freundii, the percentage of detection was same for both the tests.
MHT and Carba NP test with the modifications were repeated. The finding of all the repeats was consistent.
| Discussion|| |
Resistance to different carbapenems in increasing globally at a high speed, leading us to a situation when virtually no antibiotic will be effective for infections caused by carbapenemase-producing bacteria. In this study, the prevalence of carbapenemase resistant bacteria was 16.18%. According to meropenem yearly susceptibility test information collection program, resistance to meropenem for clinical isolates of K. pneumoniae increased significantly from 0.6% in 2004 5.6% in 2008. High prevalence of resistance to carbapenems has been reported from different parts of India., In a study done by Taneja et al., resistance of P. aeruginosa to carbapenems was 42%. High prevalence of carbapenem resistance was reported in Klebsiella spp. (31%–51%), Pseudomonas spp.(39%–59%), and E. coli (2%–13%), isolated from wards and ICU of a tertiary hospital in Delhi ranging between 13% and 59%. Another study by Gupta et al. showed the prevalence of carbapenem resistance in different GNB ranging from 17% to 22%, with maximum resistance observed in Pseudomonas spp.(30%–37.6%) and minimum in E. coli (2.1-3.5%). in a report from Vellore, based on the detection of respiratory isolates found lower resistance (12.2%) to carbapenems in nonfermenting GNB. Most of the resistance to carbapenems in our institute were observed in K. pneumoniae (62.12%), followed by E. coli (16.67%) and P. aeruginosa (10.61%). The prevalence of carbapenem resistance (16.18%) in this study was lower than most of the published reports.,, This finding can be attributed to the remote location of our institute, preventing the spread of carbapenemase-producing bacteria from the far away metro cities with high prevalence of carbapenem resistance.
In this study, MHT and Carba NP test were performed individually without knowing the genotype of the strains. Among the strains which showed resistance to one of the carbapenems, 86.4% and 75% were positive for Carba NP test and MHT, respectively. Hence, more detection of Carba NP test than MHT was observed with regard to various carbapenemases. Some carbapenemase producers were missed by MHT which were detected in Carba NP test. The sensitivity of Carba NP test among genotypically confirmed carbapenemase producers was 72.5% in a study by Tijet et al. Sensitivity of MHT was 77.4% among genotypically known carbapenemase producers in a study from India. The finding of our study has the positive results for Carba NP test (86.4%) and MHT (75%) near to these studies, and the result correlates with the study by Castanheira et al., who concluded that MHT may not be a useful screening test for detection of carbapenemases as many MBL-producing isolates could not be detected by this test. A total of 14.39% (19/132) GNB were MHT negative but Carba NP test positive. This finding may be attributed to GNB strains producing MBL though this can only be proved by molecular techniques. However, there were 4 strains which were MHT positive but Carba NP test negative. This finding can be attributed to GNB strains producing OXA-48-like carbapenemases as observed by Tijet et al.
Detection of carbapenemase producers has epidemiological significance for the prevention of further spread of carbapenem resistance in the society, which depends mostly on early detection of carriers producing these carbapenemases. Therefore, this study can be helpful for early detection of carbapenem resistance and their transmission. The gold standard for the identification of the enzymes for resistance to carbapenems is based on use of molecular techniques, mostly polymerase chain reaction (PCR)., Newer modifications of the Carba NP test  can also help identify these enzymes. It was a limitation in this study that the enzymes for resistance to carbapenems could not be identified and differentiated.
| Conclusion|| |
Detection of carbapenemase-producing bacteria has great impact on hospital infection control and for epidemiological purpose for preventing further transmission of resistance. In this study, it was shown that the Carba NP is a rapid test where the results can be read in short time. This test shows visible color change leading to simple interpretation. It is easy to perform in clinical laboratory, no costly reagent or special instrument required, and no special skill or training required. Among the limitations of Carba NP test, it can be mentioned that standardization regarding the concentration of reagents is necessary for proper interpretation and reproducibility. Inability to perform the recent modification of the test  and molecular analysis of the strains by PCR would have enhanced the quality of this study, and can be mentioned as a limitation. In future, further analysis based on molecular techniques is needed for validation of the results of this study.
We would like to thank all the staff of Department of Microbiology, Bankura Sammilani Medical College, for their invaluable support in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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