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Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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2015 (English)In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 3, p. 315-324Article in journal (Refereed) Published
Abstract [en]

Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and Lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.

Place, publisher, year, edition, pages
2015. Vol. 17, no 3, p. 315-324
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:umu:diva-103727DOI: 10.1016/j.jmoldx.2015.01.005ISI: 000353843900011PubMedID: 25772704OAI: oai:DiVA.org:umu-103727DiVA, id: diva2:818922
Available from: 2015-06-09 Created: 2015-05-28 Last updated: 2018-06-07Bibliographically approved
In thesis
1. Improved diagnosis and prediction of community-acquired pneumonia
Open this publication in new window or tab >>Improved diagnosis and prediction of community-acquired pneumonia
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Förbättrad diagnostik och prediktion vid samhällsförvärvad pneumoni
Abstract [en]

Community-acquired pneumonia (CAP) is a major cause of morbidity and mortality worldwide. Although there is wide variation in the microbial etiology, CAP may manifest with similar symptoms, making institution of proper treatment challenging. Therefore, etiological diagnosis is important to ensure that correct treatment and necessary infection control measures are instituted. This provides a challenge for conventional microbial diagnostic methods, typically based on culture and direct antigen tests. Moreover, existing molecular biomarkers have poor prognostic value. Few studies have investigated the global metabolic response during infection and virtually nothing is known about early responses after the start of antimicrobial treatment. The aim of this work was to improve diagnostic and predictive methods for CAP.

In paper I, a qPCR panel targeting 15 pathogens known to cause CAP was developed and evaluated. It combined identification of bacterial pathogens and viruses in the same diagnostic platform. The method proved to be robust and the results consistent with those obtained by standard methods. The panel approach, compared to conventional, selective diagnostics, detected a larger number of pathogens. In Paper II, whole blood samples from 65 patients with bacteremic sepsis were analyzed for metabolite profiles. Forty-nine patients with symptoms of sepsis, but later attributed to other diagnoses, were matched according to age and sex and served as a control group. Six metabolites were identified, all of which predicted growth of bacteria in blood culture. One of the metabolites, myristic acid, alone predicted bacteremic sepsis with a sensitivity of 100% and a specificity of 95%. Paper III and IV were based on a clinical study enrolling 35 patients with suspected CAP in need of hospital care. The aim was to study the metabolic response during the early phase of acute infection. The qPCR panel developed in Paper I was used to obtain the microbial etiological diagnosis. Paper IV focused on the global metabolic response and highlighted the dynamics of changes in major metabolic pathways during early recovery. A specific metabolite pattern for M. pneumoniae etiology was found. Four metabolites accurately predicted all but one patient as either M. pneumoniae etiology or not. Paper III looked at phospholipid levels during the first 48 hours after hospital admission. It was found that all major phospholipid species, especially the lysophosphatidyl-cholines, were pronouncedly decreased during acute infection. Levels started to increase the day after admission, reaching statistical significance at 48 hours. Paper II-IV showed that metabolomics might be used to study a number of different aspects of infection, such as etiology, disease progress and recovery. Knowledge of the metabolic profiles of patients may not only be utilized for biomarker discovery, as proposed in this work, but also for the future development of targeted therapies and supportive treatment.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2018. p. 80
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1960
Keywords
Community-acquired pneumonia, infection, diagnosis, qPCR, metabolites, metabolomics
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-147064 (URN)978-91-7601-873-6 (ISBN)
Public defence
2018-05-25, Bergasalen (Q0), Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-05-04 Created: 2018-04-25 Last updated: 2018-06-09Bibliographically approved

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Edin, AliciaGranholm, SusanneKoskiniemi, SatuAllard, AnnikaSjöstedt, AndersJohansson, Anders

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