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)

Abstract

BackgroundBacterial 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.MethodsBAL 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.ResultsWe evaluated 53 BAL samples to identify the bacterial pathogen and key resistance determinants. Thirty-one samples yielded growth of ≥1 × 104 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 × 104 CFU/mL of the same pathogen by culture (100% sensitivity). The MB-PCR also identified blaTEM-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.ConclusionMultiplex 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 grantAstellas: 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