Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes.

BACKGROUND: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. METHODS: Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. RESULTS: The sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. CONCLUSIONS: Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

The influence of smoking on oral neutrophils and matrix metalloproteinase-8 in periodontitis patients before and after nonsurgical treatment.

Objective: To evaluate and compare the oral neutrophil numbers (ONN) in saliva, the level of matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid (GCF) and the periodontal parameters in smokers versus non-smokers with periodontitis, before and after nonsurgical periodontal treatment (NSPT). Materials and method: 40 chronic periodontitis patients including 20 smokers and 20 non-smokers were enrolled in this quasi-experimental study. All patients were received the NSPT included instructing oral hygiene, scaling and root planing. At baseline (T0) and after NSPT 1 month (T1) and 3 months (T3), all patients were assessed for salivary ONN, GCF MMP-8, and clinical parameters like plaque index (PlI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment loss (CAL). The differences between the two groups were analyzed using the independent sample t-test and the Mann-Whitney U test; and the differences between T0, T1 and T3 of each group were analyzed with paired-samples t-test and Wilcoxon signed-rank test. The level of significance was set at 0.05. Results: The ONN was significantly less in smokers than in non-smokers although there was no significant difference in other parameters between the two groups at baseline (p > 0.05). All clinical periodontal parameters reduced significantly after 1 month and 3 months of NSPT in both groups (p < 0.01). PPD of non-smokers was significantly lower than those of smokers at T1 and T3. ONN and MMP-8 level showed a significant decrease in non-smoking subjects, while there was no significant difference in smoking ones after NSPT (T1 and T3). At 1 month after treatment, ONN tended to reduce in non-smokers whereas to increase in smokers significantly. Conclusion: Smoking reduced ONN, impaired treatment effect in reducing PPD, and changed the MMP-8 level in gingival crevicular fluid to NSPT. Trial registration: Identifier NCT04974502 in CLinicalTrials.gov.