Rapid detection of four non-fermenting Gram-negative bacteria directly from cystic fibrosis patient's respiratory samples on the BD MAX™ system

Abstract

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patient's respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.

Duplex real-time PCR assay for the simultaneous detection ofAchromobacter xylosoxidansandAchromobacterspp

“…to the genus level; however, this method currently cannot attain reliable species-level resolution, with e.g. A. xylosoxidans unable to be differentiated from A. denitrificans (18), despite these species being genetically distinct (Figure 1).…”

“…We chose the real-time PCR platform for Ac-Ax assay development due to its multiplexing capability, low per-sample cost, high accuracy potential, direct detection from polymicrobial specimens (e.g. sputum), excellent sensitivity, greater accessibility in lower-resourced laboratories, and rapid (<1h) turnaround time (18). The upfront equipment cost of real-time PCR equipment (∼USD$25-40K) is also considerably less than VITEK ® MS (USD$200K) or many next-generation sequencing platforms such as Illumina, and has a much smaller laboratory footprint.…”

“…However, all Achromobacter spp. are allocated as A. xylosoxidans / A. denitrificans on this platform (18), thus providing limited capacity for accurate species-level identification. Further, VITEK ® MS and other mass spectrometry-based platforms require a purified isolate to obtain an accurate speciation result, which limits the utility of this platform as it cannot be used directly on polymicrobial clinical specimens such as sputum, resulting in longer turn-around times, potentially incorrect antimicrobial treatment, and higher costs (19, 20).…”

“…In addition, mass spectrometry-based equipment has a large upfront cost and footprint, rendering this method out-of-reach for smaller, less-resourced laboratories. To address this shortcoming, an automated multiplex PCR has recently been developed to detect four non-fermentative Gram negative bacterial species, including A. xylosoxidans , directly from respiratory samples using the BD MAX™ System (18). This multiplex assay detected A. xylosoxidans with 97% specificity, but only 78% sensitivity (18), indicating suboptimal diagnosis of this organism using this method.…”

“…To address this shortcoming, an automated multiplex PCR has recently been developed to detect four non-fermentative Gram negative bacterial species, including A. xylosoxidans , directly from respiratory samples using the BD MAX™ System (18). This multiplex assay detected A. xylosoxidans with 97% specificity, but only 78% sensitivity (18), indicating suboptimal diagnosis of this organism using this method. Further, the BD MAX™ multiplex assay was not designed to identify other Achromobacter spp., meaning that ∼50% of CF infections caused by Achromobacter spp.…”