Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry

Abstract

The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (P<0.01). Detection of potentially pathogenic microorganisms by PCR/ESI-MS was evaluated in comparison with conventional culture-based or molecular methods. The agreement between positive findings was overall good. Most Staphylococcus aureus-positive PCR/ESI-MS results were confirmed by culture or species-specific PCR (27/33, 82%). The identity of Streptococcus pneumoniae could however be confirmed for only 6/17 (35%) PCR/ESI-MS-positive samples. Non-cultivable and fastidious pathogens, which were not covered by standard culture procedures were readily detected by PCR/ESI-MS, including Legionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients.

Comparison of Unyvero P55 Pneumonia Cartridge, in-house PCR and culture for the identification of respiratory pathogens and antibiotic resistance in bronchoalveolar lavage fluids in the critical care setting

“…Microbiological investigation of lower respiratory tract infection in patients in the critical care setting would benefit from new methodologies with increased sensitivity and turnaround time. The aetiology of pneumonia is broad and few commercial respiratory panel assays are available with a sufficiently wide range of targets, particularly for the Gram-positive and Gram-negative bacteria seen frequently in ventilator and hospital-associated pneumonia [3,4,[15][16][17]. In addition to this, rapid antibiotic resistance detection should enable more appropriate antibiotic selection to improve patient management, earlier antibiotic de-escalation to aid stewardship and earlier infection control interventions [18].…”

“…Many respiratory viral and atypical bacterial panel assays are commercially available and offer rapid molecular detection for both high-and low-throughput laboratory and near-patient test settings [1,2]. However, rapid, automated commercial molecular assays for the typical respiratory bacteria targeted by most broad-spectrum empirical antibiotic regimens are significantly fewer in number and lack important antibiotic resistance gene targets [3,4]. Therefore, antibiotic de-escalation and pathogen-targeted antimicrobial therapy for severe pneumonia identified in the intensive care unit (ICU) still relies on the results of slow, traditional microbiological culture methods.…”

Evaluation of the Biofire Filmarray Pneumonia panel plus for lower respiratory tract infections

“…Recent years' development in molecular diagnostic methods has led to a markedly increased availability of both commercial and in-house developed diagnostic panels targeting viruses and atypical bacterial pathogens [3]. However, examples of panels targeting typical respiratory bacteria are few [4][5][6], and molecular panels intended for LRTI diagnostics are even more scarce [7][8][9][10]. Methods combining targets for typical bacterial pathogens, atypical bacteria and viruses have so far been limited to qPCR-based panels, requiring a separate sample extraction step [8][9][10], which limit their use to larger clinical microbiology laboratories during working hours.…”

Emerging strategies for the noninvasive diagnosis of nosocomial pneumonia

“…In a study of more than 1000 bacterial isolates, MALDI-TOF MS showed a sensitivity of 95% and a specificity of 84.1% for sample identification compared with conventional systems, taking 6 min/isolate for identification at a cost of 22%-32% less than that of current methods [ [49] and for the detection of biological markers of inflammation in pneumonia [50]. Two studies by the same group evaluated the diagnostic performance of the IRIDICA PCR/ESI-MS assay with BAL samples from ventilated patients with suspected pneumonia [51,52] and showed promising results. By contrast, Huttner et al [53] documented a concordance of 45% between PLEX-ID and standard diagnostic methods for bacterial and fungal detection in BAL specimens.…”