Rapid detection of Burkholderia cepacia complex carrying the 16S rRNA gene in clinical specimens by recombinase-aided amplification

Abstract

The Burkholderia cepacia complex (BCC) is a group of opportunistic pathogens, including Burkholderia cepacia, Burkholderia multivorans, Burkholderia vietnamiensis and Burkholderia ambifaria, which can cause severe respiratory tract infections and lead to high mortality rates among humans. The early diagnosis and effective treatment of BCC infection are therefore crucial. In this study, a novel and rapid recombinase-aided amplification (RAA) assay targeting the 16S rRNA gene was developed for BCC detection. The protocol for this RAA assay could be completed in 10 min at 39°C, with a sensitivity of 10 copies per reaction and no cross-reactivity with other pathogens. To characterize the effectiveness of the RAA assay, we further collected 269 clinical samples from patients with bacterial pneumonia. The sensitivity and specificity of the RAA assay were 100% and 98.5%, respectively. Seven BCC-infected patients were detected using the RAA assay, and three BCC strains were isolated from the 269 clinical samples. Our data showed that the prevalence of BCC infection was 2.60%, which is higher than the 1.40% reported in previous studies, suggesting that high sensitivity is vital to BCC detection. We also screened a patient with B. vietnamiensis infection using the RAA assay in clinic, allowing for appropriate treatment to be initiated rapidly. Together, these data indicate that the RAA assay targeting the 16S rRNA gene can be applied for the early and rapid detection of BCC pathogens in patients with an uncharacterized infection who are immunocompromised or have underlying diseases, thereby providing guidance for effective treatment.

Development of a real-time recombinase-aided amplification assay for rapid and sensitive detection of Edwardsiella piscicida

“…Compared with the general molecular detection methods, RAA possesses several advantages, such as low-cost, simplicity of operation (as it does not necessitate complex equipment or highly skilled personnel, can be conducted using portable device), low and constant reaction temperature (39°C), and rapid reaction speed (within 20 min) ( Xie et al., 2012 ). As a result, RAA has been widely used in identifying viruses, bacteria, and parasites ( Cui et al., 2022 ; Fu et al., 2022 ; Lin et al., 2022 ; Cui H et al., 2023 ; Cui X et al., 2023 ). Consequently, it represents a rapid and user-friendly method suitable for clinical use, particularly in primary laboratories.…”

“…Previous studies on viral and bacterial pathogens have indicated the detection limit of the RAA assay varied from 10 to 142 copies per reaction when tested against standard plasmids ( Zhang et al., 2017 ; Cui et al., 2022 ; Fu et al., 2022 ; Lin et al., 2022 ; Cui H et al., 2023 ; Cui X et al., 2023 ). In this study, a novel and simple method for E. piscicida detection was proposed based on RAA technique.…”

Burkholderia Cepacia: An Emerging Superbug in Intensive Care Unit Settings of Tertiary Care Hospitals in Pakistan

“…This was contrary to a study by Patra et al in 2014 in which susceptibility to Meropenem was 100%, followed by Ceftazidime-sulbactam and Pipera-cillin Tazobactam. 17 Another study in Bangladesh showed 100% sensitivity to Meropenem, and 93% of isolates were resistant or intermediate to levofloxacin. 19 The results from SENTRY Antimicrobial surveillance program showed greater than 90% susceptibility to Minocycline, similar to our results in which Minocycline is considered the most effective antibiotic.…”

“…A study published in the Annals of Tropical Medicine and Health showed that the prevalence of Burkholderia cepacia was highest from blood cultures. 17 A study in China showed that respiratory specimens were on top. 18 Our study demonstrated that Minocycline was the least resistant antimicrobial agent, followed by Cotrimoxazole 6.2%, Chloramphenicol 7.15% and Levofloxacin 11%.…”

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions

“…RPA can amplify targets up to 1.5 kb but is better suited to amplicons between 100 and 200 bp, with an optimal amplification time of 20 min [ 9 , 11 , 13 ]. Recently, RPA has successfully targeted Burkholderia spp., including Burkholderia pseudomallei [ 14 , 15 , 16 ], Burkholderia mallei [ 17 ], and BCC [ 18 ]. More recently, Fu et al [ 18 ] reported a recombinase-aided amplification (RAA) targeting the 16S rRNA gene for BCC detection.…”