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

Discussion

The timely implementation of antibiotic treatment according to bacterial identification and antibiotic resistance results is important in improving the outcome of septic patients.[29] The BC-GN assay is a microarray-based test in which blood culture broth can be directly used to detect the most common pathogenic gram-negative bacteria and their resistance determinants. This is the first Asian multicenter study to compare rapid testing methods on direct blood cultures, namely the microarray-based BC-GN assay and MALDI-TOF MS. This study presents results concordant with previous BC-GN evaluation data for the identification of targeted gram-negative bacteria, with concordance rates of 99.1%. In addition, the BC-GN results presented excellent identification of polymicrobial samples with 100% correct identification. In addition, one sample presented as polymicrobial in BC-GN, whereas in conventional culture, only one bacterium was identified. Therefore, for the identification of polymicrobial samples, BC-GN might be more sensitive than conventional culture for some samples. Misidentification of nontargeted bacterial species occurred, with three wrongly identified bacteria among 13 nontargeted bacteria. This high analytical sensitivity and specificity is consistent with several previous studies that have shown the high accuracy of BC-GN assay for direct blood cultures, with generally more than 95% agreement for the identification of targeted gram-negative bacteria.[3–12,30]

Meanwhile, MALDI-TOF MS has been conventionally used for the identification of bacteria in colonies after subculture. Recently, studies have been conducted to show the usefulness of MALDI-TOF MS for the direct identification of bacteria from blood cultures with significant turnaround time reduction.[13–27] These studies used several different specimen preparation protocols, including the lysis-centrifugation method,[13] saponin method,[15,17] serum separator method,[31] identification after subculture of 3 to 5 hours,[25] and various other modifications.[27] In these studies, the correct identification rate of gram-negative bacteria varied between 80% and 90%. In our study, the concordance rate for the identification of monomicrobial cultures was 86.2%; therefore, it was not significantly different from the results of previous studies. In previous studies, MALDI-TOF MS presented high positive predictive values, such that bacteria were not detected or were correctly identified but rarely misidentified. In our study, there was no misidentification; however, MALDI-TOF MS could not identify bacteria with low identification scores and reported "no reliable id." In previous studies on polymicrobial cultures, MALDI-TOF MS identified either one bacterial species or none of the species, and all the species were correctly identified in a minor proportion of 10% to 20% of samples.[13,15–17,24,31] We evaluated the mixed analysis mode of MALDI-TOF MS. By using the mixed mode, we could correctly identify two gram-negative bacteria in one of five (20%) of polymicrobial cultures, and in the remaining four polymicrobial samples, one major bacterium was identified. Compared with the BC-GN assay, MALDI-TOF MS is more cost effective and not limited to the targeted species. However, specimen handling is more complicated and identification rates are lower, especially for polymicrobial cultures.

The other important clinical use of the BC-GN assay is for the detection of antimicrobial resistance determinants simultaneously with bacterial identification. The production of CTX-M-type enzymes is the most prevalent resistance mechanism of ESBL.[32] It has been reported that although CTX-M-type ESBLs have been detected in many gram-negative bacteria, the main clinically important ESBLs are E coli and K pneumoniae.[32] In our study, BC-GN assay detected blaCTX-M in more than half of K pneumoniae cultures and in approximately 30% of E coli. and Proteus species cultures. Approximately 80% of bacteria positive for blaCTX-M presented cefotaxime and ceftazidime resistance by antibiotic susceptibility testing after pure culture. BC-GN assay also detected five important carbapenem-resistance determinants. Among the included bacterial species, most of the Acinetobacter baumannii samples had blaOXA; therefore, the bacteria were imipenem resistant. In addition, more than half of K pneumoniae samples were detected as CRE, with blaNDM being the most frequently detected, and some samples had blaKPC; most of them (93%) were also resistant to imipenem. In 20% of Citrobacter species, blaNDM was found. Therefore, by using the BC-GN assay, a substantial proportion of CRE could be screened except for a few cases. In a previous study, P aeruginosa cultures were found to be imipenem resistant without the detection of carbapenem resistance determinants by BC-GN assay.[5] In our study, one P aeruginosa culture was imipenem resistant with the BC-GN-detected resistance determinants. Meanwhile, previous studies have concluded that bacteria that test positive for carbapenem-resistance determinants by BC-GN should be considered CRE.[5] Therefore, in our study, some cultures were detected to have CRE despite appearing to be imipenem susceptible by conventional culture. In addition, resistance markers could be detected when nontargeted bacteria were present in the culture. In our study, a culture with Aeromonas caviae presented bla CTX-M positivity. Even without bacterial identification, the early detection of antimicrobial resistance may help antimicrobial stewardship in clinical practice.

A previous study investigating the clinical impact of BC-GN assay has demonstrated that using this assay for patient management significantly reduces the length of intensive care unit stay and mortality.[3] We evaluated the possible impact of BC-GN assay on reducing turnaround time in a simulated clinical setting. Because it may not be possible to perform BC-GN assay immediately when the blood culture is positive due to a high burden of labor time and cost, we simulated the reduction of turnaround time when BC-GN assay was performed in the daytime on weekdays. Using this strategy, the turnaround time was significantly reduced in comparison with the reporting time using conventional culture. This reduction was more prominent for polymicrobial samples.

Our study showed that BC-GN assay presented superior performance compared with MALDI-TOF MS assay for the identification of gram-negative bacteria in direct blood cultures for targeted gram-negative bacteria, especially for polymicrobial specimens. BC-GN assay had a unique clinical impact, as it could detect important antibiotic resistance determinants that MALDI-TOF MS could not detect. However, considering the limit of targeted bacteria and more expensive laboratory costs, it may be impetuous to conclude that BC-GN assay is superior to MALDI-TOF MS. A reasonable laboratory testing strategy using either BC-GN assay or MALDI-TOF MS according to the distribution of bacteria and antibiotic resistance could improve patient outcomes, decrease the cost and burden on clinical laboratories, and facilitate more rapid and clinically useful reporting.

processing....