Abstract
Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.
Highlights
According to the World Health Organization (WHO), lower respiratory tract infections (LRTIs) were the main cause of morbidity and mortality leading to 3 million deaths worldwide in 2016 [1]
LRTIs fall into two categories based on their origin: community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) [4]
The four pathogens selected for the RB4 multiplex real-time polymerase chain reaction (mRT-polymerase chain reaction (PCR)) assay, K. pneumoniae, P. aeruginosa, S. aureus, and M. catarrhalis, are species with clinical significance and were detected at 1.3– 24.7%, 0–8%, 0.4–10.4%, 0.3–15%, respectively, from pneumonia patients [5]. yphG and regA were selected for the detection of K. pneumoniae and P. aeruginosa, respectively, because they have been evaluated with great sensitivity in the literature [16, 17]
Summary
According to the World Health Organization (WHO), lower respiratory tract infections (LRTIs) were the main cause of morbidity and mortality leading to 3 million deaths worldwide in 2016 [1]. LRTIs, caused by various bacteria and viruses, are associated with different clinical symptoms and etiologies affected by age, sex, and season [2]. Owing to complicated clinical symptoms, efforts to reduce the global burden of LRTIs using preventive and treatment strategies require timely identification of pathogens [3]. In the Asia-Pacific region, 11 species of bacteria and eight species of viruses causing CAP are examined by serology, culture, immunofluorescence assay, or polymerase chain reaction (PCR) [5]. In LRTI patients with severe CAP, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa are the most common species, accounting for 28.6%, 28.6%, and 17.9%, respectively [6], of infections and are identified by diagnostic culture methods. Despite low incidence, Moraxella catarrhalis has been recently recognized as an important emerging pathogen because it shows prevalent resistance to some antibiotics through beta-lactamase production [7]
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