Rapid quantification of Staphylococcus aureus from endotracheal aspirates of ventilated patients: a proof-of-concept study

Abstract

Major concern for intubated patients is ventilator-associated pneumonia (VAP). Early detection of VAP and its causative microorganism(s) is a key challenge for clinicians. Diagnosis is based on clinical, radiological, and microbiological elements, the latter being provided 24-48h after sampling. According to practices, clinicians can sample endotracheal aspirates (ETAs) so as to check for patient colonization or perform ETA in case of VAP suspicion. In this proof-of-concept study, we report the evaluation of a semiautomated molecular method to rapidly quantify Staphylococcus aureus, one of the most involved microorganisms in VAP, directly from raw ETA samples. After evaluation using artificial ETA samples, our method was applied on 40 clinical ETA samples. All S. aureus-positive samples were successfully detected and quantified. Our method can provide an efficient sample preparation protocol for all raw ETA samples, combined with an accurate quantification of the bacterial load, in less than 3h 30min.

Benefit-to-risk balance of bronchoalveolar lavage in the critically ill. A prospective, multicenter cohort study

“…Undoubtedly, if there is one alternative to the BAL that could be less risky for immunosuppressed patients, it is the combination of non-invasive tests on sputum, nasopharyngeal secretions, urine, and blood that may dramatically restrict the number of patients for whom BAL is absolutely needed [12]. This might become increasingly true in view of the current development of molecular diagnostic tools [28][29][30], the knowledge acquired in chest highresolution computed tomography imaging [31,32], and perhaps future progress in the human volatilome analysis [33].…”

Molecular quantification of bacteria from respiratory samples in patients with suspected ventilator-associated pneumonia

“…Each BAL specimen (500 mL) was incubated with 5 mg lysin (Hyglos GmbH, Bernried am Starnberger See, Germany) for 15 min at 37 C before nucleic acid extraction. For ETA specimens, liquefaction and pre-processing were performed as previously described [7]. DNA was extracted from all lysates using the NucliSENS ® easyMag ® (bioM erieux SA, Marcy l'Etoile, France), according to the specific B2.0.1 protocol, with an elution volume of 55 mL.…”

“…Primers and probes were used to detect the SPC and the main pathogens involved in VAP (see Supplementary material, Quantifications were performed using qPCR standard curves, by converting Ct to CFU/mL. Briefly, for each pathogen, 100 mL of a calibrated McFarland solution of a reference strain (representing a bacterial quantity of approximately 10 7 CFU and determined precisely by plating dilutions) was transferred into a tube containing 400 mL 0.9% NaCl for BAL standards, or 300 mL of a solution previously described [7]. These reference strains were either obtained from an external provider (ATCC, LGC Standards, Molsheim, France) or from bioM erieux's internal biobank.…”

“…On the contrary, for P. aeruginosa and H. influenzae, qPCR had better concordance results than conventional culture (89.5% (66.9%e (a) Quantified loads of each specimen (Log 10 scale) were plotted for both methods (culture method considered as reference standard method in x-axis and molecular method in y-axis).The number of represented specimens is written between brackets for aggregated specimens (same loads measured by both methods). Regarding traditional culture, the results outside the quantification limits (<10 3 or >10 7 ) are expressed as equal to 0 or 10 7 Overall percentage agreements (per cent of patients with either positive or negative quantification in both specimens, using a threshold at 10 4 CFU/mL for BAL and 10 6 CFU/mL for ETA) were 91.2% (for molecular method) and 91.2% (for standard method) for S. aureus, and 98.1% (for molecular method) and 94.1% (for standard method) for P. aeruginosa.…”

Ultra-Sensitive and Specific Detection of S. aureus Bacterial Cultures Using an Oligonucleotide Probe Integrated in a Lateral Flow-Based Device

“…These techniques are precise and effective; however, they are time consuming, provide delayed information (2 to 3 days), and are expensive due to the need of trained personnel and specific lab equipment. Nowadays, PCR-based approaches and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) methods are being used for their speed and sensitivity [3][4][5][6][7][8][9]. However, amplification methods like PCR suffer from contamination problems, difficult experimental design, complex interpretation of results, and high costs.…”