Microarray-Based Nucleic Acid Assay and MALDI-TOF MS Analysis for the Detection of Gram-Negative Bacteria in Direct Blood Cultures

Seon Young Kim, MD, PhD; Jeong Su Park, MD, PhD; Yun Ji Hong, MD, PhD; Taek Soo Kim, MD, PhD; Kiho Hong, MD, PhD; Kyoung-Ho Song, MD, PhD; Hyunju Lee, MD, PhD; Eu Suk Kim, MD, PhD; Hong Bin Kim, MD, PhD; Kyoung Un Park, MD, PhD; Junghan Song, MD, PhD; Sun Hoe Koo, MD, PhD; Eui-Chong Kim, MD, PhD

Disclosures

Am J Clin Pathol. 2019;151(2):143-153. 

In This Article

Materials and Methods

Study Design and Sample Preparation

We tested 124 blood culture samples that were positive for various gram-negative bacteria collected from four tertiary hospitals (Seoul National University Bundang Hospital, Seoul National University Hospital, Seoul Medical Center, and Chungnam National University Hospital) from December 2014 to October 2015. Among 124 specimens, 82 random samples were collected into BacT/ALERT FAN aerobic and anaerobic culture bottles (bioMérieux, Marcy l'Etoile, France), and 42 samples were collected into BACTEC Plus Aerobic/F and Anaerobic/F culture bottles (Becton Dickinson, Sparks, MD). The culture bottles were incubated in a BacT/ALERT 3D system (bioMérieux) or BACTEC FX system (Becton Dickinson) until the samples were positive for microbial growth, according to the manufacturer's instructions. This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB number B-1404-248-301).

BC-GN Test Procedure and Interpretation of Results

For a total of 124 samples, BC-GN tests were performed according to the manufacturer's protocol. When the culture system reported growth of bacteria, Gram staining was performed. Then, 1 mL of each blood culture was transferred to a tube and centrifuged at 3,000 rpm for 2 minutes. The resultant supernatants (350 μL) were transferred to a tube for the BC-GN assay. The 350-μL blood culture medium samples were transferred to an extraction tray, and standard procedures were followed. After the reaction was completed, microarray images were captured and interpreted, and the results are reported according to the system's interpretation algorithm. The BC-GN microarray contains hexaplicate spots for each target as well as several control spots, including two internal controls—IC1 (process control) and IC2 (hybridization control)—and negative and background control spots.

MALDI-TOF MS

Out of the 124 total samples, 100 were tested in MALDI-TOF MS assay, as this assay was incorporated into the study after the start of the BC-GN assay. The MALDI-TOF MS assay was performed as follows: when the growth of bacteria was detected by the culture system, 5 mL of blood culture medium was transferred to a serum separation tube and centrifuged at 1,500g for 1 minute. Of the resultant supernatant, 1.5 mL was transferred to a tube and centrifuged at 13,200 rpm for 1 minute. After the supernatant solution was removed, the pellet was washed with distilled water. The washed pellet was used for the MALDI-TOF MS assay. The pellet was spotted onto a target plate of Microflex LT (Bruker Daltonics, Bremen, Germany), and the test was performed according to the manufacturer's instructions.[28] Mass spectra were analyzed using MALDI Biotyper reference library version 3.3.1.2 (Bruker).

Identification and Susceptibility Testing After Subculture

For all included samples, bacterial subculture of positive blood culture media was performed on 5% sheep blood agar plates at 37°C in 5% CO2 for 18 to 24 hours. Conventional biochemical identification and microdilution antibiotic testing of isolates from pure cultures were performed using the MicroScan WalkAway 96 Plus system (Siemens Healthcare Diagnostics, West Sacramento, CA) or Vitek2 system (bioMérieux).

Statistical Analysis

Data are presented as number and percentage for categorical variables and median and range for continuous variables. Concordance rates were calculated for the BC-GN assay results compared with the final identification and susceptibility results for the subcultured microbial isolates. Median times to detection by culture and BC-GN were compared using the Mann-Whitney U test. Statistical analyses were conducted using SPSS version 20.0 software (SPSS, Chicago, IL). The statistical significance level was set as P <.05.

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