This was a prospective study done in the Department of Microbiology, SVS Medical College from July 2018-December 2018. Ethical clearance certificate (SVSMC/IEC Approval/No.05/2018-623) was taken from the institutional ethical committee. All gram negative bacterial isolates from patients admitted in the hospital were included and all the gram positive isolates and samples isolates from outpatient were excluded from the study. A total of 612 Gram negative organisms were isolated during the study period. Clinical samples such as pus, blood, sputum, stool, urine, endotracheal aspirates and other body fluids were taken-up for the study. Samples were identified up to the species level and antibiotic sensitivity testing was done by Vitek 2 systems as per standard protocols which has usual sensitivity of 99% and specificity of 96% [11]. All the carbapenem resistant organisms isolated were further tested for carbapenemase production. The carbapenemase production was tested using both commercially available RAPIDEC Carba NP test (Biomerieux) as per the standard protocols and an in-house Carba NP test.

For the in-house carba NP test, two 1.5 mL Eppendorf tubes were taken for each strain and labeled as A and B. 200 μL of sterile water was added to tube A and emulsified with 2-3 loop full of bacterial colony to make a heavy suspension. From this, 100 μL of the inoculum was transferred to tube B. Separately, Solution A (0.05% phenol red, 10 mM ZnSO₄, pH 7.8) and Solution B (1 mL sol A, 6 mg imipenem powder) were prepared. Now, 100 μL of sol A was added to tube marked A and 100 μL of sol B was added to tube B and both the tubes were incubated at 37°C for 2 hours. A change in colour from red to yellow was considered as positive Carba NP test indicating carbapenemase production [10].

Twenty five carbapenem sensitive isolates were also included for the analysis. The positive and negative controls used were Klebsiella pneumoniae ATCC BAA-1705 and Klebsiella pneumonia ATCC BAA-1706.

Culture of the clinical samples → identification of the organism → detection of carbapenem resistance → detection of carbapenemase production by RAPIDEC carba NP + in-house Carba NP test → result obtained in 2 hours.

Statistical analysis was performed by SPSS VERSION 20 and graph pad prism software version 6.0. The collected data was entered in to MS excel and tabulated. Sensitivity, specificity, positive predictive and negative predictive values were detected.

A total 612 Gram negative organisms were isolated during the study period, out of which 91 isolates were identified as carbapenem resistant and were further tested for carbapenemase production by both our in-house Carba NP test as well as RAPIDEC Carba NP test [Table/Fig-1] Out of the 91 carbapenem resistant strains tested, 72 were identified as carbapenemase producers in both the methods. Out of them, Klebsiella pneumoniae accounted for 59.7% of the total carbapenemase producing organisms followed by Pseudomonas aeruginosa (25%) and Escherichia coli (6.9%). Acinetobacter and Enterobacter constituted less than 5% of the strains showing carbapenemase production [Table/Fig-2]. Among Enterobacteriaceae, 43 out of 59 carbapenem resistant Klebsiella were carbapenemase producers whereas all the 5 carbapenem resistant E.coli were positive for Carba Np test. Among Pseudomonas, 18 out of 21 isolates were carbapenemase producers. The performance of the test in detecting carbapenemase production was high when considering the enterobacteriaceae members like E.coli and Klebsiella where as it was low for non-fermenters like Acinetobacter. Out of 72 carbapenemase producing organisms, 44 positive cases are from males and 28 are from females as shown in [Table/Fig-3].

[Table/Fig-1]:

Results from both in house Carba NP test (left side) and RAPIDEC carba NP (Biomerieux) (right side) showing positive for carbapenemase production.

[Table/Fig-2]:

Distribution of carbapenemase producing bacterial isolates in the present study.

[Table/Fig-3]:

Gender wise distribution of the isolates obtained and those that tested positive for carbapenemase production in the present study.

All the positive isolates identified by our in-house Carba NP test were also positive by commercial RAPIDEC test and none of the 25 carbapenem sensitive strains tested were positive for carbapenemase production by either method indicating 100% correlation between the two methods studied.

The overall sensitivity of the test was 79.12% (CI:69.33% to 86.94%) and specificity was 82.79% (CI: 71.48% to 85.14%). The positive predictive value PPV was 82.91% (CI:71.48% to 85.14%). and negative predictive value NPV was 79.38% (CI:71.65% to 94.08%).

[Table/Fig-4] shows the distribution of carbapenemase positive Gram negative bacteria from various clinical samples. Klebsiella pneumoniae was the predominant carbapenemase producer and most of the carbapenemase producing GNB were isolated from endotracheal secretions, followed by blood and others.

Multidrug resistance is increasingly being observed both in community acquired and hospital acquired pathogens. Carbapenems are the drugs of choice in majority of the serious nosocomial infections. With the wide spread emergence of carbapenem resistance especially in enterobacteriaceae members like Klebsiella, it has become extremely difficult for the clinicians to treat such cases as only few alternatives like tigecycline and colistin are available for the treatment [12]. As the antibiotic pipeline is almost empty, it is mandatory that the emergence of resistance to carbapenems is contained by early identification and adherence to appropriate infection control measures along with the best clinical practices. Sound knowledge about the mechanisms of carbapenem resistance and identification methods will help in achieving this goal. As carbapenemase production is the predominant mechanism for resistance to carbapenems, early detection of carbapenemase producing organisms is extremely important in preventing the spread of these infections.

Nordmann P et al., developed a novel method for rapid detection of these carbapenemase producers [5]. The basic principle of this test was the hydrolysis of carbapenem by the bacterial enzyme carbapenemase which detects the pH changes by measuring the change of the colour of phenol red indicator. Based on this principle, many commercial kits have been developed among which RAPIDEC Carba NP test developed by Biomerieux is widely used with good sensitivity and specificity [10,13,14]. Our in-house Carba NP test was also based on the same principle.

In the present study, both in-house Carba NP and commercial RAPIDEC Carba NP tests were evaluated in their ability to detect carbapenemase producing isolates. Out of the total 91 carbapenem resistant isolates tested, 72 were identified to be positive for carbapenemase production by both the tests. In addition, none of the carbapenem sensitive isolates tested were positive by either test. Klebsiella was the most common isolate showing carbapenemase production. Gupta V et al., tested 75 bacterial strains and found all strains to be positive for carbapenemase production by both Carba NP and RAPIDEC Carba NP tests [10]. They also observed Klebsiella as the most common bacterial isolate showing carbapenemase production in their study. But they have also used other methodologies like Modified Carba NP (MCNP) test, Carbapenem Inactivation Method (CIM) test. Similarly, Dortet L et al.. compared RAPIDEC Carba NP, the Rapid CARB Screen and the Carba NP test and found that sensitivity and specificity were 99% and 100%, respectively for the RAPIDEC Carba NP test, 96.8% and 100% for the Carba NP test [15]. They have stated that RAPIDEC Carba NP possesses the best performance for rapid and efficient detection of carbapenemase-producing Enterobacteriaceae and suggested it as a first-line screen of carbapenemase-producing Enterobacteriaceae in clinical settings. Comparatively, limited literature is available in India against western countries with regards to the utilisation of rapid carbapenemase detection methods [16-18]. Wide utilisation of these rapid detection methods in a country like India will help not only in decreasing the mortality and morbidity but also the health care economical burden.

RAPIDEC carba NP test was extremely useful in the early detection of carbapenemase producing isolates in our hospital because of the rapid result which can be obtained within 2 hours and also doesn’t needed any technical expertise. Because of this early detection, appropriate infection control practices could be placed which further decreased the spread of these infections. The high cost of RAPIDEC carba NP test may limit its use routinely in clinical laboratories in low settings but the inhouse Carba NP test is a viable option for its routine use in such settings as it is quite cheap and easy to perform and interpret.

Though various methodologies are available for the detection of carbapenemases, the methods should be individualised in accordance with the needs, work load and economical constraints of the hospitals. Nevertheless, rapid detection of these infections plays a major role in the appropriate management of the patients and initiation of infection control measures thus preventing the spread of these infections.

The limitation of this study, is it has less sensitivity towards OXA-48 carbapenemases. No genotyping of these isolates was done to identify any resistance mechanisms underlying.

Both the Carba NP in-house as well as commercial RAPIDEC Carba NP test were equally effective in the identification of the carbapenemase producers among the Gram negative bacilli. Wide adaptability of these tests by various laboratories will help in the early identification of these potentially spreading carbapenemase producers, which in association with appropriate treatment and infection control practices will prevent the emergence and decreases the mortality and morbidity associated with these bacterial infections.

Declaration: Author had presented the Abstract (Oral Presentation) in International Conference held at Manipal, Karnataka, India.