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Quantitative detection of bacterial DNA in whole blood in bloodstream infection
Örebro University, School of Medical Sciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis aims to increase the knowledge on how quantitative PCR can be used in the diagnostics of bloodstream infections, with an emphasis on quantitative elements.

In Papers I and II, we evaluated quantitative data from two commercial PCR tests for pathogen detection directly in blood, Magicplex Sepsis (I) and SeptiFast (II), from patients with suspected sepsis. We found that high quantification cycle (Cq) values, indicating low DNA loads, were associated with findings of pathogens with doubtful clinical relevance, whereas low Cq values, indicating high DNA loads, were correlated with sepsis and septic shock, as well as with positive blood culture results.

In Paper III, we aimed to study the bacterial DNA load during Staphylococcus aureus bacteremia, in relation to different clinical factors. For this purpose, we developed a droplet digital PCR (ddPCR) for precise DNA quantification, targeting S. aureus specifically. We found that a high initial S. aureus DNA load was associated with laboratory markers for immune dysregulation as well as with sepsis, endocarditis, and mortality.

In Paper IV, we aimed to develop a tool for repeated DNA quantification during bloodstream infection. For this purpose, we optimized a ddPCR, targeting the universal bacterial 16S rDNA, and performed a comparison with species-specific ddPCRs on spiked blood, and on clinical samples. The performance of the16S rDNA ddPCR was adequate, and we found that a high 16S rDNA load was associated with sepsis and mortality.

In conclusion, our results indicate that the pathogen DNA load in blood plays an important role in the clinical picture in BSI. In future research on molecular BSI diagnostics, studies on DNA loads and clearance should be included.

Place, publisher, year, edition, pages
Örebro: Örebro University , 2018. , p. 94
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 183
Keywords [en]
Bloodstream infection, bacteremia, sepsis, DNA load, quantitative PCR, droplet digital PCR, 16S rDNA
National Category
General Practice
Identifiers
URN: urn:nbn:se:oru:diva-68452ISBN: 978-91-7529-257-1 (print)OAI: oai:DiVA.org:oru-68452DiVA, id: diva2:1238817
Public defence
2018-10-05, Örebro universitet, Campus USÖ, hörsal C1, Södra Grev Rosengatan 32, Örebro, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2018-08-14 Created: 2018-08-14 Last updated: 2018-09-13Bibliographically approved
List of papers
1. Evaluation of a Commercial Multiplex PCR Assay for Detection of Pathogen DNA in Blood from Patients with Suspected Sepsis
Open this publication in new window or tab >>Evaluation of a Commercial Multiplex PCR Assay for Detection of Pathogen DNA in Blood from Patients with Suspected Sepsis
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167883Article in journal (Refereed) Published
Abstract [en]

The Magicplex Sepsis Real-time Test (MST) is a commercial multiplex PCR that can detect more than 90 different pathogens in blood, with an analysis time of six hours. The aim of the present study was to evaluate this method for the detection of bloodstream infection (BSI). An EDTA whole blood sample for MST was collected together with blood cultures (BC) from patients with suspected sepsis at the Emergency Department of a university hospital. Among 696 study patients, 322 (46%) patients were positive with at least one method; 128 (18%) were BC positive and 268 (38%) were MST positive. Considering BC to be the gold standard, MST had an overall sensitivity of 47%, specificity of 66%, positive predictive value (PPV) of 23%, and a negative predictive value of 87%. Among the MST positive samples with a negative BC, coagulase-negative staphylococci (CoNS) and species that rarely cause community-acquired BSI were frequently noted. However, the quantification cycle (Cq) values of the MST+/BC- results were often high. We thus hypothesized that the performance of the MST test could be improved if the Cq cut-off level was adjusted downwards. With a lower Cq cut-off value, i.e. 6.0 for Staphylococcus species and 9.0 for all other species, the number of MST positive cases decreased to 83 (12%) and the overall sensitivity decreased to 38%. However, the PPV increased to 59% and the specificity increased to 96%, as many MST positive results for CoNS and bacteria that rarely cause community-acquired BSI turned MST negative. In conclusion, our study shows that with a lower Cq cut-off value, the MST will detect less contaminants and findings with unclear relevance, but to the cost of a lower sensitivity. Consequently, we consider that a positive MST results with a Cq value above the adjusted cut-off should be interpreted with caution, as the result might be clinically irrelevant. In a correspondent way, quantitative results could probably be useful in the interpretation of positive results from other molecular assays for the detection of BSI.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library of Science, 2016
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:oru:diva-54404 (URN)10.1371/journal.pone.0167883 (DOI)000392842900017 ()27997618 (PubMedID)2-s2.0-85006866953 (Scopus ID)
Note

Funding Agency:

Resaerch committee of Region Örebro Län

Available from: 2017-02-07 Created: 2017-01-10 Last updated: 2018-09-12Bibliographically approved
2. Quantitative data from the SeptiFast real-time PCR is associated with disease severity in patients with sepsis
Open this publication in new window or tab >>Quantitative data from the SeptiFast real-time PCR is associated with disease severity in patients with sepsis
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2014 (English)In: BMC Infectious Diseases, ISSN 1471-2334, E-ISSN 1471-2334, Vol. 14, no 1, article id 155Article in journal (Refereed) Published
Abstract [en]

Background: The commercial test, SeptiFast, is designed to detect DNA from bacterial and fungal pathogens in whole blood. The method has been found to be specific with a high rule-in value for the early detection of septic patients. The software automatically provides information about the identified pathogen, without quantification of the pathogen. However, it is possible to manually derive Crossing point (Cp) values, i.e. the PCR cycle at which DNA is significantly amplified. The aim of this study was to find out whether Cp values correlate to disease severity.

Methods: We used a study cohort of patients with positive results from SeptiFast tests for bacteria from a recent study which included patients with suspected sepsis in the Emergency department. Cp values were compared with disease severity, classified as severe sepsis/septic shock or non-severe sepsis, according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine.

Results: Ninety-four patients were included. The prevalence of severe sepsis/septic shock in the study was 29%. SeptiFast positive tests from patients with severe sepsis/septic shock had significantly lower Cp values compared with those from patients with non-severe sepsis, median 16.9 (range: 7.3 - 24.3) versus 20.9 (range: 8.5 - 25.0), p < 0.001. Positive predictive values from the SeptiFast test for identifying severe sepsis/septic shock were 34% at Cp cut-off <25.0, 35% at Cp cut-off <22.5, 50% at Cp cut-off <20.0, and 73% at Cp cut-off <17.5. Patients with a positive Septifast test with a Cp value <17.5 had significantly more severe sepsis/septic shock (73% versus 15%, p < 0.001), were more often admitted to the Intensive Care Unit (23% versus 4%, p = 0.016), had positive blood culture (BC) more frequently (100% versus 32%, p < 0.001) and had longer hospital stays (median 19.5 [range: 4 - 78] days versus 5 [range: 0 - 75] days, p < 0.001) compared with those with a Cp value >17.5.

Conclusions: Our results suggest that introducing quantitative data to the SeptiFast test could be of value in assessing sepsis severity. Moreover, such data might also be useful in predicting a positive BC result.

Place, publisher, year, edition, pages
London: BioMed Central, 2014
Keywords
Polymerase-chain-reaction, blood-stream infections, staphylococcus-aureus bacteremia, genomic bacterial load, rapid detection, united-states, pathogens, diagnosis, culture, epidemiology
National Category
Infectious Medicine
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-34951 (URN)10.1186/1471-2334-14-155 (DOI)000333601600004 ()24656148 (PubMedID)2-s2.0-84899124239 (Scopus ID)
Note

Funding Agency:

Research Committee of the Örebro County Council

Available from: 2014-05-05 Created: 2014-05-05 Last updated: 2018-09-12Bibliographically approved
3. nuc DNA in Whole blood in Relation to Immune dysregulation, Sepsis, and Mortality in Staphylococcus aureus Bacteremia
Open this publication in new window or tab >>nuc DNA in Whole blood in Relation to Immune dysregulation, Sepsis, and Mortality in Staphylococcus aureus Bacteremia
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(English)Manuscript (preprint) (Other academic)
National Category
General Practice
Identifiers
urn:nbn:se:oru:diva-68880 (URN)
Available from: 2018-09-12 Created: 2018-09-12 Last updated: 2018-09-12Bibliographically approved
4. 16S rDNA droplet digital PCR for monitoring bacterial DNAemia in bloodstream infections
Open this publication in new window or tab >>16S rDNA droplet digital PCR for monitoring bacterial DNAemia in bloodstream infections
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(English)Manuscript (preprint) (Other academic)
National Category
General Practice
Identifiers
urn:nbn:se:oru:diva-68881 (URN)
Available from: 2018-09-12 Created: 2018-09-12 Last updated: 2018-09-12Bibliographically approved

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