Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens

Benson, Robert F.; Tondella, M. Lucia; Bhatnagar, Julu; Carvalho, Maria da Glória; Sampson, Jacquelyn S.; Talkington, Deborah F.; Whitney, Anne M.; Mothershed, Elizabeth A.; McGee, Lesley; Carlone, George M.; McClee, Vondguraus; Guarner, Jeannette; Zaki, Sherif R.; Dejsiri, Surang; Cronin, Kassi; Han, Jian; Fields, Barry S.

doi:10.1128/jcm.01858-07

Cited by 40 publications

(27 citation statements)

References 14 publications

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“…Clinicians resort to empiric antibiotics up front and often arbitrary continuation or de-escalation of antibiotics subsequently, resulting in antibiotic abuse and the emergence of multidrug-resistant bacteria [2,7,8,24]. Greater use of PCR amplification for bacteria may allow more precise antibiotic prescription, and routine use of multiplex PCR kits for viruses [8,20,45,46,47,48] could provide answers regarding aetiology. However, as specific treatments such as antivirals for respiratory viruses other than influenza do not yet exist, the true utility of these PCR kits remains unclear [10].…”

Section: Discussionmentioning

confidence: 99%

The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia

Siow

Koay²,

Lee³

et al. 2016

Respiration

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Background: Pathogens are often not identified in severe community-acquired pneumonia (CAP), and the few studies using polymerase chain reaction (PCR) techniques for virus detection are from temperate countries. Objective: This study assesses if PCR amplification improves virus and bacteria detection, and if viral infection contributes to mortality in severe CAP in a tropical setting, where respiratory pathogens have less well-defined seasonality. Methods: In this cohort study of patients with severe CAP in an intensive care unit, endotracheal aspirates for intubated patients and nasopharyngeal swabs for non-intubated patients were sent for PCR amplification for respiratory viruses. Blood, endotracheal aspirates for intubated patients, and sputum for non-intubated patients were analysed using a multiplex PCR system for bacteria. Results: Out of 100 patients, using predominantly cultures, bacteria were identified in 42 patients; PCR amplification increased this number to 55 patients. PCR amplification identified viruses in 32 patients. In total, only bacteria, only viruses, and both bacteria and viruses were found in 37, 14, and 18 patients, respectively. The commonest viruses were influenza A H1N1/2009 and rhinovirus; the commonest bacterium was Streptococcus pneumoniae. Hospital mortality rates for patients with no pathogens, bacterial infection, viral infection, and bacterial-viral co-infection were 16.1, 24.3, 0, and 5.6%, respectively (p = 0.10). On multivariable analysis, virus detection was associated with lower mortality (adjusted odds ratio 0.12, 95% confidence interval 0.2-0.99; p = 0.049). Conclusions: Viruses and bacteria were detected in 7 of 10 patients with severe CAP with the aid of PCR amplification. Viral infection appears to be independently associated with lower mortality.

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Section: Discussionmentioning

confidence: 99%

The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia

Siow

Koay²,

Lee³

et al. 2016

Respiration

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“…115 Another commercially available BBMA e has been used for the detection of 6 respiratory bacterial pathogens including Streptococcus pneumoniae, Neisseria meningitidis, encapsulated or nonencapsulated Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydia pneumoniae. Although this assay had less sensitivity when compared to real-time PCR, it was useful for surveillance purposes 15 and, when combined with a commercial assay including viral respiratory pathogens f for a 21-plex assay, significant coinfection rates could be established. 22,25 In-house developed assays have also been used 97,98 to detect Norovirus, Rotavirus, and Sapovirus species, as well as astroviruses and adenoviruses from feces.…”

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confidence: 99%

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

et al. 2013

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Abstract. Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

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“…However, this conventional method is time consuming, laborious, and less sensitive [4, 5]. There are a lot of factors that may influence the culture results, including antimicrobial treatment, prolonged transportation time, and inappropriate transportation medium [5, 6]. Due to these limitations, the need to utilize more sensitive methods such as multiplex PCR, real-time PCR, and microarray assays are now greatly increased.…”

Section: Discussionmentioning

confidence: 99%

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Khazani

Zuraina

Yean

et al. 2017

Journal of Tropical Medicine

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Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

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Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens

Cited by 40 publications

References 14 publications

The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia

The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

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