Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents

Tosun A. I., Kolukirik M., Yılmaz M., Ötgün S. N., AYGÜN G., Kolukirik C. Z. K., ...Daha Fazla

Journal of Microbiological Methods, cilt.206, 2023 (SCI-Expanded) identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 206
  • Basım Tarihi: 2023
  • Doi Numarası: 10.1016/j.mimet.2023.106690
  • Dergi Adı: Journal of Microbiological Methods
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, EMBASE, Environment Index, Food Science & Technology Abstracts, MEDLINE, Veterinary Science Database
  • Anahtar Kelimeler: Carbapenem-resistant Enterobacteriaceae, Hospital-acquired infection, Methicillin-resistant Staphylococcus aureus, Real-time PCR, Vancomycin-resistant Enterococcus
  • İstanbul Üniversitesi Adresli: Evet

Özet

Aims: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. Methods: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). Results: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. Conclusions: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.