Evaluation of a Multiplex PCR Assay with Molecular Beacon Probes to Rapidly Detect Bacterial Pathogens Directly in Bronchial Alveolar Lavage (BAL) Samples from Patients with Hospital-Acquired Pneumonia (HAP)

BACKGROUND: Bacterial pneumonia is a common complication in hospitalized patients and it is associated with high morbidity and mortality. Standard culture-based methods may take 2–3 days to identify the etiologies of HAP, leading to delays in appropriate therapy. A rapid molecular assay that could diagnose the etiology of bacterial pneumonia directly from BAL samples within a few hours could facilitate faster and more directed administration of antimicrobial therapy. METHODS: BAL samples were collected from hospitalized patients with suspected pneumonia, including ventilated patients, from December 2016 through April 2017. Genomic DNA was isolated from BAL samples using NucliSENS(®) easyMAG(®). A panel of target-specific molecular beacon probes in a real time PCR assay (MB-PCR) was used to identify the following pathogens: universal bacterial Identification (16S rRNA), E. coli (uidA), K. pneumoniae (gapA), S. aureus (spa), P. aeruginosa (rpsL), A. baumannii (Ab-ITS) and the following resistance determinants: ESBLs (CTX-M, TEM and SHV), carbapenemases (NDM, VIM, IMP, OXA-48 and KPC) and mecA. The results of MB-PCR were then compared with quantitative culture results performed by the clinical microbiological lab. RESULTS: We evaluated 53 BAL samples to identify the bacterial pathogen and key resistance determinants. Thirty-one samples yielded growth of ≥1 × 10(4) CFU/mL of bacteria by quantitative culture. The bacterial identification using MB-PCR for 16S rRNA correctly identified the presence of bacteria in all 31 samples (100% sensitivity). The MB-PCR identified P. aeruginosa (n = 5), S. aureus (n = 5), E. coli (n = 1), A. baumannii (n = 1), and K. pneumoniae (n = 1) in BAL samples that yielded ≥1 × 10(4) CFU/mL of the same pathogen by culture (100% sensitivity). The MB-PCR also identified bla(TEM)-harboring E. coli that grew ampicillin-resistant E. coli by culture. The specificity of the16S rRNA probe was 70%, as 7/53 BAL were false positive, whereas the specificity for the MB-PCR was 100% for P. aeruginosa, S. aureus, E. coli, and A. baumannii, and 98% for K. pneumoniae. CONCLUSION: Multiplex MB-PCR assay is a rapid, sensitive and specific tool for detection of common bacterial causes of nosocomial pneumonia and important resistance determinants directly from BAL samples. DISCLOSURES: M. Satlin, Hardy Diagnostics: Investigator, Research support; S. G. Jenkins, Cormedix: Consultant, Consulting fee; Bayer: Consultant, Consulting fee Merck: Grant Investigator and Scientific Advisor, Research grant; T. J. Walsh, The Medicines Company: Consultant and Investigator, Consulting fee and Research grant Astellas: Consultant and Investigator, Consulting fee and Research grant Allergan: Consultant and Investigator, Consulting fee and Research grant Merck: Consultant and Investigator, Consulting fee and Research grant