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

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.

[1]  Lei Huang,et al.  Genetic Diversity and Pathogenic Features in Klebsiella pneumoniae Isolates from Patients with Pyogenic Liver Abscess and Pneumonia , 2022, Microbiology spectrum.

[2]  B. Kan,et al.  Development and evaluation of a sensitive recombinase aided amplification assay for rapid detection of Vibrio parahaemolyticus , 2022, Journal of Microbiological Methods.

[3]  Hengyi Xu,et al.  The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7. , 2021, Molecular and cellular probes.

[4]  Yan Zhou,et al.  Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay , 2021, Frontiers in Microbiology.

[5]  Lin Gan,et al.  A Recombinase Aided Amplification Assay for Rapid Detection of the Klebsiella pneumoniae Carbapenemase Gene and Its Characteristics in Klebsiella pneumoniae , 2021, Frontiers in Cellular and Infection Microbiology.

[6]  J. Lipuma,et al.  Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products , 2021, Pathogens.

[7]  Jin-ding Chen,et al.  Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus , 2021, Life.

[8]  J. Dennis,et al.  Advances in Phage Therapy: Targeting the Burkholderia cepacia Complex , 2021, Viruses.

[9]  J. Sherchand,et al.  Biofilm Formation by Pathogens Causing Ventilator-Associated Pneumonia at Intensive Care Units in a Tertiary Care Hospital: An Armor for Refuge , 2021, BioMed research international.

[10]  Jian Li,et al.  Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus , 2021, Frontiers in Cellular and Infection Microbiology.

[11]  Qingping Wu,et al.  Development of a recombinase-aided amplification assay for rapid detection of human norovirus GII.4 , 2020, BMC Infectious Diseases.

[12]  Wenjing Wang,et al.  Recombinase-aided amplification–lateral flow dipstick assay—a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus , 2020, Poultry science.

[13]  J. Degrossi,et al.  Distribution of Burkholderia cepacia complex species isolated from industrial processes and contaminated products in Argentina , 2020, International Microbiology.

[14]  J. Bao,et al.  Clinical Validation of Two Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of African Swine Fever Virus , 2020, Frontiers in Microbiology.

[15]  C. Cerniglia,et al.  A comparison of culture-based, real-time PCR, droplet digital PCR and flow cytometric methods for the detection of Burkholderia cepacia complex in nuclease-free water and antiseptics , 2020, Journal of Industrial Microbiology & Biotechnology.

[16]  I. Sá-Correia,et al.  Burkholderia cepacia Complex Bacteria: a Feared Contamination Risk in Water-Based Pharmaceutical Products , 2020, Clinical Microbiology Reviews.

[17]  A. Horsley,et al.  Antibiotic treatment for Burkholderia cepacia complex in people with cystic fibrosis experiencing a pulmonary exacerbation. , 2020, The Cochrane database of systematic reviews.

[18]  Jing Yuan,et al.  Use of a rapid recombinase-aided amplification assay for Mycoplasma pneumoniae detection , 2020, BMC Infectious Diseases.

[19]  M. V. von Specht,et al.  Burkholderia cepacia complex: 11 years of surveillance in patients with Cystic Fibrosis in Posadas, Argentina. , 2019, Revista Argentina de microbiologia.

[20]  V. Waters,et al.  Microbiology of Cystic Fibrosis Airway Disease , 2019, Seminars in Respiratory and Critical Care Medicine.

[21]  Z. Zhan,et al.  Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7 , 2019, Virology Journal.

[22]  Xinxin Shen,et al.  A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction , 2019, BMC Infectious Diseases.

[23]  M. Sfeir Burkholderia cepacia complex infections: More complex than the bacterium name suggest. , 2018, The Journal of infection.

[24]  E. Mylonakis,et al.  The Cost-Effectiveness of Rapid Diagnostic Testing for the Diagnosis of Bloodstream Infections with or without Antimicrobial Stewardship , 2018, Clinical Microbiology Reviews.

[25]  Saijun Tang,et al.  Rapid detection of Salmonella with Recombinase Aided Amplification. , 2017, Journal of microbiological methods.

[26]  Megha Sharma,et al.  MALDI-TOF mass spectrometry: An emerging tool for unequivocal identification of non-fermenting Gram-negative bacilli , 2017, The Indian journal of medical research.

[27]  H. Schweizer,et al.  Antibiotic resistance in Burkholderia species. , 2016, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[28]  P. Vandamme,et al.  Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers , 2016, Applied Microbiology and Biotechnology.

[29]  G. A. Whitmore,et al.  Clinical and demographic factors associated with post-lung transplantation survival in individuals with cystic fibrosis. , 2015, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[30]  Jacqueline A. Keane,et al.  Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins , 2014, Nucleic acids research.

[31]  J. Goldberg,et al.  Recurrent Burkholderia infection in patients with chronic granulomatous disease: 11-year experience at a large referral center. , 2009, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[32]  P. Dřevínek,et al.  Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia , 2008, BMC Microbiology.

[33]  J. Govan,et al.  Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples , 2007, Journal of Clinical Microbiology.

[34]  L. Kalish,et al.  Impact of Burkholderia dolosa on lung function and survival in cystic fibrosis. , 2006, American journal of respiratory and critical care medicine.

[35]  Sylvain Brisse,et al.  Multilocus Sequence Typing of Klebsiella pneumoniae Nosocomial Isolates , 2005, Journal of Clinical Microbiology.

[36]  M. Whiteford,et al.  Outcome of Burkholderia (Pseudomonas) cepacia colonisation in children with cystic fibrosis following a hospital outbreak. , 1995, Thorax.