Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-Producing Enterobacteriaceae from Bacterial Colonies and Directly from Positive Blood Cultures

Abstract

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is critical for appropriate treatment and infection control. We compared a rapid fluorogenic assay using a carbapenem-based fluorogenic probe with other phenotypic assays: modified carbapenem inactivation method (mCIM), Carba NP test (CNP), and carbapenemase inhibition test (CIT). A total of 217 characterized isolates of Enterobacteriaceae were included as follows: 63 CPE; 48 non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae (non-CP-CRE); 53 extended-spectrum β-lactamase producers; and 53 third-generation-cephalosporin-susceptible isolates. The fluorogenic assay using bacterial colonies (Fluore-C) was conducted by lysing the isolates followed by centrifugation and mixing the supernatant with fluorogenic probe. In addition, for the fluorogenic assay using spiked blood culture bottles (Fluore-Direct), pellets were obtained via the saponin preparation method, which can directly identify the pathogens using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The fluorescence signal was measured over 50 min using a fluorometer. The fluorescent signal of CPE was significantly higher than that of non-CPE in both Fluore-C (median relative fluorescence units [RFU] [range], 5,814 [240 to 32,009] versus 804 [36 to 2,480], respectively; P < 0.0001) and Fluore-Direct (median RFU [range], 10,355 [1,689 to 31,463] versus 1,068 [428 to 2,155], respectively; P < 0.0001) tests. Overall, positive and negative percent agreements of Fluore-C, mCIM, CNP, CIT, and Fluore-Direct were 100% and 98.7%, 98.3% and 97.5%, 88.1% and 100%, 96.4% and 98.7%, and 98.3% and 98.1%, respectively. The relatively lower positive percent agreement (PPA) of CNP was mainly observed in OXA-type CPE. The fluorogenic assay showed excellent performance with bacterial colonies and also directly from positive blood cultures. We included many non-CP-CRE isolates for strict evaluation. The fluorogenic assay will be a useful tool for clinical microbiology laboratories.

Aplikasi Antimicrobial Susceptibility Test (AST) dalam Mendeteksi Carbapenem-Resistant Enterobacteriaceae (CRE): Sebuah Tinjauan Sistematis

“…Dari sebelas studi yang ditelaah, hampir separuh studi (45,4%), yaitu Sri Lanka, Singapura, China, dan Korea Selatan. [10][11][12][13][14] Lokasi studi pada umumnya dilakukan di rumah sakit umum, tiga studi dilakukan di rumah sakit pendidikan dan dua studi dilaku-J Indon Med Assoc, Volum: 73, Nomor: 3, Juni -Juli 2023 kan di laboratorium universitas. Berdasarkan tujuan studi yang ditelaah, prevalensi CRE didapatkan sebesar 9,6% pada studi skrining kasus CRE.…”

“…5 Sedangkan studi-studi lain yang bertujuan mendeteksi CP-CRE menunjukkan prevalensi CRE yang lebih bervariasi, yakni mulai dari 3,8% hingga 100%. 10,[12][13][14][15][16][17][18][19][20] Mayoritas sampel berasal dari urin (27,2%) dan darah (27,2%). Sebagian kecil sampel berasal dari usap dubur, sekret trakea, ujung kateter, cairan asites, usap luka, tinja, sputum, selang endotrakeal, usap vagina, cairan peritoneal, cairan purulen prapankreas, dan abses perianal.…”

“…Dari sembilan studi yang mencantumkan metode AST, Vitek-2 ® adalah metode yang paling sering digunakan untuk mengidentifikasi CRE (n=5), kemudian diikuti disk diffusion (n=3). 10,11,13,14,[16][17][18][19][20] Sementara itu, dua studi lainnya hanya mencantumkan jumlah kasus positif CRE tanpa menyebutkan metode AST yang digunakan dalam mendeteksi CRE. 12,15 Kelima studi tersebut kebanyakan mendeteksi carbapenemase-producing CRE (CP-CRE) dengan metode polymerase chain reaction (PCR) dan carba Nordmann-Poirel (carba NP) (Tabel 1).…”

Hindsight Is 2019—the Year in Clinical Microbiology

“…MBLs require zinc for activity, and zinc is chelated by EDTA; this distinction is important due to differences in therapeutic options [19]. Several studies evaluated using modified carbapenem (or other β-lactam) inactivation assays directly from blood culture bottles with good results [20][21][22][23].…”

Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice

“…Recently, a lot of Phenotypic detection methods of CRE has been developed, such as Nitro Speed-CarbaNP Test ( Nordmann et al., 2020 ), Rapidec CarbaNP ( Elawady et al., 2019 ), CarbaNP-Direct test ( Banu et al., 2019 ), MALDI-TOF MS rapid detection method ( Oho et al., 2021 ), Fluorogenic Assay method ( Kim et al., 2020 ) and so on. A large proportion of methods were very complex or need very expensive instrument and aren’t fit the use of common clinical laboratory.…”