Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

Abstract

A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH 4 HCO 3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers. Nosocomial infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas spp. are now emerging worldwide and are difficult to treat (1). Previous data have shown that strict epidemiological intervention based on the rapid detection of carbapenemase production can prevent the spread of those bacteria (2). Therefore, the introduction of rapid and sensitive methodologies for the detection of carbapenemase-producing bacteria is of utmost importance. Recently, two new highly sensitive and rapid methods for the direct detection of carbapenemase activity were developed. In 2011, two research groups demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can detect carbapenemase activity, based on mass spectral profiles obtained from carbapenem molecules (3, 4). The second method, the Carba NP test, is a biochemical method used in the detection of carbapenemase activity in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter species (5-7). This test is based on a decrease in pH resulting from the hydrolysis of the ␤-lactam ring of carbapenem molecules, which is detected using phenol red as a pH indicator.The aim of this study was to validate the efficiency of a MALDI-TOF MS meropenem hydrolysis assay and the Carba NP test for the detection of carbapenemase producers. The performances of both methods were compared with that of a recently published modification of the MALDI-TOF MS assay (MALDI-TOF BIC) in an effort to increase the reliability of detecting OXA-48 producers (8). Furthermore, the automatic interpretation of all methods was evaluated.The methods were tested against a group of 124 Enterobacteriaceae and 37 Pseudomonas aeruginosa isolates from collections of the Faculty of Medicine and University Hospital in Plzen (Czech Republic), the National Medicines Institute in Warsaw (Poland), the Robert Koch Institute in Wernigerode (Germany), and the University Hospital in Larissa (Greece). The isolates were previously characterized, as described below. All isolates were tested for extended-spectrum ␤-lactamase (ESBL) and AmpC expression by the ESBL double-disk synergy test (DDST) with cefotaxime, ceftazidime, aztreonam, and amoxicillin-clavulanate disks in the absence and presence of cloxacillin (250 g/ml) (9). Susceptibility to carbapenems was determined by using imipenem, merope...

The Problem of Carbapenemase-Producing-Carbapenem-Resistant-Enterobacteriaceae Detection

“…Like the Carba NP test, MALDI-TOF assays have reported difficulty detecting OXA-48-like enzymes. One study reported a sensitivity of 77% and a specificity of 100%, with the lower sensitivity being due to missing 16 of 19 OXA-48 producers (24). However, this report suggested that the MALDI-TOF carbapenemase assay could be modified (by the addition of bicarbonate) to improve its performance for the OXA-48 producers with little impact on detection of other enzymes (sensitivity improved to 98%) (24).…”

“…Imipenem hydrolysis changes the pH and produces a resultant color change of a pH indicator. The Carba NP is faster than the MHT described above and has excellent sensitivity for most carbapenemases (reported range, 73 to 100%) (18,23,24,25). Its sensitivity for the OXA-48 carbapenemase is much lower; only 1 of 17 OXA-48 producers was detected in one study (24).…”

“…The Carba NP is faster than the MHT described above and has excellent sensitivity for most carbapenemases (reported range, 73 to 100%) (18,23,24,25). Its sensitivity for the OXA-48 carbapenemase is much lower; only 1 of 17 OXA-48 producers was detected in one study (24). Similar tests based on the same principle have also been developed, including the Blue-Carba test, the Rosco Rapid Carb screen, and the Rapidec Carba NP test (26,27,28,29).…”

“…One study reported a sensitivity of 77% and a specificity of 100%, with the lower sensitivity being due to missing 16 of 19 OXA-48 producers (24). However, this report suggested that the MALDI-TOF carbapenemase assay could be modified (by the addition of bicarbonate) to improve its performance for the OXA-48 producers with little impact on detection of other enzymes (sensitivity improved to 98%) (24). Use of the MALDI-TOF assay for carbapenemase detection is a technique that typically requires the use of alternate MALDI-TOF instrument settings (on the usual instrument) than those used for FDA-approved microbial identification.…”

“…MALDI-TOF MS detects specific carbapenem degradation products when bacterial protein extracts are incubated with carbapenems (24,32). Like the Carba NP test, MALDI-TOF assays have reported difficulty detecting OXA-48-like enzymes.…”