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High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia
Keiko Hamano-Hasegawa1, Miyuki Morozumi, Eiichi Nakayama
1Kitasato Institute for Life Sciences, Laboratory of Molecular Epidemiology for Infectious Agents, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
Abstract:
We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.
Insights
A new real-time RT-PCR method accurately detects 13 respiratory viruses and six bacteria in pediatric community-acquired pneumonia (CAP). This diagnostic advance aids in identifying coinfections and guides appropriate antimicrobial use.
Area of Science:
- Medical Microbiology
- Virology
- Pediatric Infectious Diseases
Background:
- Community-acquired pneumonia (CAP) in children presents diagnostic challenges.
- Accurate identification of viral and bacterial pathogens is crucial for effective treatment.
- Coinfections with multiple pathogens can complicate diagnosis and management.
Purpose of the Study:
- To develop and validate a multiplex real-time RT-PCR assay for simultaneous detection of common respiratory viruses and bacteria.
- To investigate the prevalence of viral and bacterial etiologies, including coinfections, in pediatric CAP cases.
Main Methods:
- Development of a real-time reverse transcription-PCR (RT-PCR) assay.
- Detection of 13 respiratory viruses (Influenza A/B, RSV A/B, PIV 1-3, adenovirus, rhinovirus, enterovirus, coronavirus OC43, hMPV, HBoV).
- Simultaneous detection of six bacterial pathogens in 1700 pediatric CAP patient samples.
Main Results:
- Etiology remained unknown in 22.6% of cases.
- Bacterial infections were suspected in 34.4% (single/multiple), viral infections in 27.9% (single/multiple), and coinfections in 15.2%.
- Key identified pathogens included Streptococcus pneumoniae (24.4%), Mycoplasma pneumoniae (14.8%), Rhinovirus (14.5%), and RSV (9.4%).
Conclusions:
- The developed multiplex RT-PCR method enhances diagnostic accuracy for pediatric CAP.
- This assay facilitates the identification of viral, bacterial, and coinfections.
- Improved diagnostics can lead to more appropriate antimicrobial prescribing practices.
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