Rapid Time to Results and High Sensitivity of the CarbaNP Test on Early Cultures

Abstract

bFor rapid results, we adapted the CarbaNP test by using 5-hour-old cultures; the sensitivity and specificity for detection of carbapenemase production were 100% for non-OXA-producing organisms. The sensitivity from early cultures was 94% for detection of carbapenemase production in Enterobacteriaceae strains. Utilizing younger cultures allows the test to be incorporated into a single day's workflow, facilitating timely patient care and antimicrobial stewardship. C arbapenemase-producing Enterobacteriaceae strains pose significant problems in clinical practice because of limited treatment options (1). Prompt detection of these organisms with current screening techniques, including the modified Hodge test (MHT), growth on selective media, and antimicrobial susceptibility testing, is difficult and time-consuming (2-5). Molecular methods are the gold standard for identification of carbapenemase genes (5, 6), but they are expensive, require technical experience, and are unable to detect novel genes (6). The CarbaNP test is inexpensive, rapid, and easy to perform and interpret; it utilizes imipenem hydrolysis, visualized by a color change, to identify carbapenemase production in Enterobacteriaceae and Pseudomonas species (7-10). In previous publications, isolates were taken from 1-day-old cultures (11) or the age of the culture was not mentioned (7-10). This study aims to determine whether the age of the isolates and the media used affect the ability of the CarbaNP test to detect carbapenemase production.A total of 157 isolates (Enterobacteriaceae, n ϭ 111; Pseudomonas spp., n ϭ 38; Acinetobacter spp., n ϭ 8) were suspected to harbor a carbapenemase because they had at least one of the following features: disc diffusion testing using CLSI guidelines (12) demonstrating decreased susceptibility to ertapenem with a zone diameter of Ͻ22 mm, meropenem MIC of Ն0.5 mg/liter determined by using a Vitek 2 system (bioMérieux Inc., Durham, NC), growth on ChromID Carba medium (bioMérieux, Inc., Durham, NC), or a positive MHT result. These isolates were recovered from clinical or environmental specimens at three tertiary hospital microbiology laboratories in Melbourne, Australia, between 2004 and 2014.All isolates underwent PCR testing using primers designed for the carbapenemase genes bla IMP , bla KPC , bla NDM , bla OXA-23-like , bla OXA-24/40-like ,bla ,bla OXA-51-like ,bla OXA-58-like ,bla OXA-48-like , and bla VIM (13-17). Fifty-eight isolates were confirmed to be carbapenemase producers by PCR, with typing as follows: IMP, n ϭ 34; NDM, n ϭ 6; KPC, n ϭ 3; VIM, n ϭ 2; OXA-23, n ϭ 6; OXA-23-like/OXA-51-like, n ϭ 1; OXA-24, n ϭ 1; OXA-181, n ϭ 2; OXA-24/40, n ϭ 1; OXA-48, n ϭ 1; OXA-51, n ϭ 1. IMP and NDM carbapenemases were subtyped by sequencing for selected isolates.The CarbaNP test was performed according to published methods (1,8,18), with modifications of culture age and inoculum. Solution A contained phenol red solution with ZnSO 4 , and solution B contained phenol red solution, ZnSO 4 and 6 mg/ml imipenem (7,8,18). Al...

Prevalence and Molecular Epidemiology of Extended-Spectrum-β-Lactamase (ESBL)-Producing Escherichia coli from Multiple Sectors of Poultry Industry in Korea

“…If MIC was >2 mg/L, the isolate was regarded as a colistin-resistant organism [ 26 ]. The CarbaNP [ 27 ] or modified carbapenem inactivation method was applied [ 28 ] if the isolates exhibited carbapenem resistance without compatible carbapenemase genes.…”

Local acquisition and nosocomial transmission of Klebsiella pneumoniae harbouring the blaNDM‐1 gene in Australia

“…Both K. pneumoniae isolates demonstrated carbapenemase activity using the Carba NP assay 1 . Molecular tests using polymerase chain reaction and sequencing for carbapenemase, extended‐spectrum β‐lactamase, plasmid‐mediated AmpC β‐lactamase and 16S ribosomal RNA methylase genes were performed as described elsewhere 2 .…”

Direct determination and differentiation of carbapenemases of A. baumannii from uncultured tracheal samples

“…Phenotypic methods like Modified Hodge Test (MHT) and disk diffusion tests with inhibitors are commonly used in microbiology laboratory to test the carbapenemase activity of isolated bacterial cultures. However, these methods show lower specificity and sensitivity compared to molecular methods [10]. Identification of the resistance gene is considered as gold standard for the confirmation of drug resistance, though they are economically and technically beyond the reach for many clinical laboratories [3, 5].…”

“…CarbaNP is a huge improvement over other phenotypic methods with almost 100% sensitivity and its specificity is comparable to molecular methods [2]. Several modifications have been made to this assay in order to improve its performance characteristics and avoid false negatives [10, 13 and 14]. Different classes of carbapenemases have been reported in gram negative pathogens.…”