Detection of carbapenemase-resistant Enterobacterales in blood culture in 15 minutes

doi:10.21203/rs.2.23537/v1

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Detection of carbapenemase-resistant Enterobacterales in blood culture in 15 minutes

https://doi.org/10.21203/rs.2.23537/v1

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Background: Bacteremia leading to sepsis and organ dysfunction is a life-threatening situation. One major challenge in treatment of sepsis is the uprising prevalence of antibiotic resistant bacteria, such as Carbapenem-resistant Enterobacterales (CRE). In recent years several molecular assays have been developed for the detection of CRE mechanisms, enabling rapid results reporting. We evaluated the performance of NG-Test CARBA 5 (NG Biotech) kit for detection of CRE, directly from patients’ blood culture.

Methods: Carbapenemase-producing (CP) CRE-positive isolates (n=38) and non-carbapenemase CRE (non-CP) isolates (n=10), previously identified using the routine methods practiced at the clinical microbiology laboratory of the Baruch Padeh Medical Center, Israel were used in this analysis. Variable concentrations of the bacterial isolates were added to a suspension composed of blood unit and saline simulating the composition of a blood culture. Samples were then transferred to an anaerobic blood culture bottle and later tested it with the NG-Test CARBA 5 (NG Biotech) kit, that identifies the CRE mechanism in 15 minutes.

Results: The NG-Test CARBA 5 kit correctly identified 43 samples (89.5%). The sensitivity and specificity of the kit were 86.8% and 100%, respectively.

Conclusions: The NG-Test CARBA 5 kit is a reliable and accessible tool for the rapid diagnosis of CRE bloodstream infections.

General Microbiology

Infectious Diseases

Bacteremia

Carbapenem-resistant Enterobacterales

NG-Test CARBA 5

Bloodstream infections

Blood culture

Bloodstream infections (BSI) are a leading cause of morbidity and mortality worldwide (1,2). Diagnosis of BSI is primarily blood culture (BC)-based, involving injection of patient blood into a bottle containing liquid culture medium, which is then placed into a designated incubator that monitors bacterial growth by means of CO₂ measurements (3). When bacterial growth is identified, the laboratory staff samples the respective BC bottle, performs Gram staining and inoculates the sample on solid agar medium, together resulting in the prolonged turnaround time of pathogen identification in BSI (at least 24–48 h, depending on the organism). Furthermore, antibiotic susceptibility testing (AST), which can only be performed after the growth of the pathogen colonies on solid medium, demands approximately 24 hours. Each delay in pathogen identification and antibiotic treatment adjustment has serious implications for patients, particularly with regards to infections with multidrug resistant bacteria such as carbapenem-resistant Enterobacterales (CRE) (4–6). Therefore, rapid assays for pathogen identification in BSI are needed.

CRE are Gram-negative bacteria that are resistant to carbapenems, a class of antibiotics used to treat severe infections, as well as to most other antibiotics commonly used today (7). CREs are classified as carbapenemase-producing CREs (CP-CREs) and non-CP-CRE subgroups, based on their ability to produce carbapenemases, the enzymes that hydrolyze the antibiotic. There are five different carbapenemase enzymes, i.e., Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), carbapenem-hydrolyzing oxacillinases (OXA-48-like), Verona integron-encoded metallo-β-lactamase (VIM) and IMP (active on imipenem) (7).

In recent years, various rapid pathogen detection assays have been developed for BC sampling (8). These methods are mainly based on molecular biology, require extensive training and dedicated equipment, and incur extra costs. Another recent development involves rapid bacterial antigen identification, which serves mainly for the identification of drug-resistant bacteria and circumvents the need for molecular methods. In particular, the NG-Test CARBA 5 kit (NG Biotech, Guipry, France) is a colourimetric assay allowing for detection of the CRE resistance mechanism, all within 15 minutes (9). Originally, the NG-Test CARBA 5 kit was developed for detection of carbapenemases in isolated colonies. Recently, the manufacturer advertised a protocol for direct detection of CRE bacteria in blood culture, without the need for bacterial cultivation (10). Several studies have evaluated the reliability of the NG Test CARBA 5 kit as a diagnostic tool for recognition of CRE in bacterial colonies (10,11). The current work assessed the kit's performance and lower limit of detection for blood CRE samples.

Bacterial isolates

This study was performed in the clinical microbiology laboratory at the Baruch Padeh Medical Center, Israel. Bacterial clinical or screening samples previously found to be resistant to carbapenems using our routine methods, were retested. Out of the 48 isolates, 10 were non-CP CRE, and 38 were CP-CRE: 1 IMP, 10 NDM, 8 OXA-48, 6 VIM, and 13 KPC. Briefly, testing involved Enterobacterales identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology (Bruker Daltonics, Bremen, Germany), and then with the β CARBA kit (Bio-Rad Laboratories Ltd., Rishon Lezion, Israel), which detects strains with a decreased susceptibility to carbapenems. Colonies that gave a positive result were further analysed using the Xpert^® Carba-R (Cepheid, Sunnyvale, CA, USA) PCR assay, that detects the five most prevalent carbapenemases (KPC, NDM, VIM, OXA-48, and IMP). In order to demonstrate that bacteria type plays no role in kit performance, different types of CRE bacteria were tested, such as Escherichia coli and Klebsiella pneumoniae. Moreover, we examined bacteria with various types of carbapenemases (NDM, KPC, OXA, VIM, IMP).

Additionally, 10 non-CP CRE isolates were collected. All isolates were stored at -80°C until analysis. Prior to analysis by the NG-Test kit, the isolates were grown on MacConkey agar (BD Diagnostics, Sparks, MD), at 36±1^oC, in 5% CO₂, for 24 h.

Blood culture preparation

Suspensions simulating patient blood samples were prepared by diluting human blood with saline, at a ratio of 45% blood unit and 55% saline. Then, a bacterial suspension of each isolate, was prepared in saline, to a turbidity of 0.5 McFarland, which is equivalent to 1-2*10⁸ CFU/ml. The bacterial suspension was then further diluted, to create variable concentrations of bacteria that were later mixed with the blood suspensions. Blood-bacteria suspensions (9 ml) were introduced to an anaerobic blood culture bottle [Plus Anaerobic/F Culture Vials (BD Diagnostics, Sparks, MD)], producing "blood cultures" with a final concentration of 6.75* 10⁷ CFU/ml. Samples from each bottle were then tested with the NG-Test CARBA 5 (NG Biotech) kit. The lowest concentration recognized by the NG Test CARBA 5 kit was determined for each pathogen tested.

Detection of carbapenemases by the NG-Test CARBA 5 kit

The NG-Test CARBA 5 kit (NG Biotech, Guipry, France) is a qualitative rapid immunoassay, in which mouse monoclonal antibodies directed against KPC (K), OXA (O), VIM (V), IMP (I), and NDM (N), are immobilized on the nitrocellulose membrane test zones K, O, V, I and N, respectively. The tested sample is mixed with 150 μL extraction buffer; 100 μL of this mixture is then dispensed into the cassette well and allowed to migrate towards the conjugate pad. If a carbapenemase is present in the suspension, it reacts with labelled monoclonal anti-carbapenemase antibodies. The carbapenemase-antibodies complex migrates through the nitrocellulose membrane and is captured by corresponding monoclonal anti-carbapenemase antibodies immobilized on the membrane, resulting in a red line (or lines) on the test zone (s) and on the control zone.

Samples from each “blood culture” (1 ml) were transferred into an Eppendorf tube, after which, 1ml lysis buffer (Accuprep Genomic DNA extraction kit, Bioneer, Korea) was added. The Eppendorf tube was vortexed for 20 sec and then centrifuged for 1 min at 13,000 rpm/min. Pellets were then washed with 1 ml wash buffer (Accuprep Genomic DNA extraction kit, Bioneer, Korea) vortexed for 20 sec, and centrifuged for 1 min at 13,000 rpm/min. Pellets were then resuspended in 150 µl extraction buffer (supplied with the kit) and placed on the test zone of the cassette and later analyzed for results.

As mentioned above, for each isolate, we tested several concentrations of isolate colonies per ml "blood culture" in order to define what is the lowest concentration that the kit can identify. It should be mentioned that each of the isolates was correctly identified by the kit, when we tested directly the isolate's colonies (without preparing a blood-bacteria suspension).

Statistical analysis

The Xpert^® Carba-R (Cepheid, Sunnyvale, CA, USA) PCR assay served as the reference method for calculating sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV). Therefore, specimens that were found positive or negative by the Xpert^® Carba-R were defined as "True Positive" or "True Negative", respectively.

Agreement between Xpert^® Carba-R and the NG-Test CARBA 5 kit was calculated as the percentage of samples that had the same results for both assays, out of the total number of tested samples. Data were analyzed using SAS® version 9.3 (SAS Institute, Cary, NC, US).

The distribution of the different bacterial isolates according to their carbapenem resistance mechanism is presented in Table 1. It should be noted that this distribution does not represent the prevalence of the different carbapenemases.

Identification of CRE mechanism in blood culture

Out of the 48 tested isolates, the NG-Test CARBA 5 kit correctly identified 43 samples (89.5%). Overall, 5 (10.5%) isolates yielded a false negative result, and no isolates generated a false positive (incorrect identification of the CRE mechanism) or an invalid result (Table 2). While the kit identified all the 11(100%) KPC-producers, 8 of the 10 (80%) NDM-producer isolates, 7 of the 8 (87.5%) OXA-48-producing bacteria and 5 of the 6 (83.3%) VIM-producers were correctly identified. The one IMP-producer isolate was not detected by the kit. Taken together, the NG-Test CARBA 5 kit's sensitivity, specificity, NPV and PPV for the detection of CRE in blood cultures were 89.6%, 100%, 66.7% and 100%, respectively (Table 3).

Determination of the average and lowest concentrations of bacteria identified by the kit

Table 4 presents the average and lowest concentrations of bacteria in blood culture identified by the NG-CARBA5 kit, for each resistance mechanism. The lowest average concentration detected by the kit was 3.57*10⁷CFU/ml, for KPC-producer isolates, while the highest average concentration was 5.37*10⁷ CFU/ml, for OXA-48-producer isolates. The lowest bacterial concentration detected was 4.12*10⁶ CFU/ml, for a KPC–producer isolate. NDM, VIM and OXA-48 producing isolates were detected at concentration of 3.33*10⁷ CFU/ml.

Detection of carbapenemase-resistant Enterobacterales in blood culture in 15 minutes

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Abstract

Introduction

Materials And Methods

Results