Evaluation of a multiplex OnSpot Primer-Extension PCR assay in the diagnosis of sepsis

Knabl, Ludwig; Mutschlechner, Wolfgang; Orth‐Höller, Dorothea

doi:10.1016/j.mimet.2015.12.001

Cited by 7 publications

(11 citation statements)

References 17 publications

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“…The broad spectrum of PAMP detection by the FcMBL ELLecSA was reflected in our ability to rapidly (< 1 h) detect > 80% of infections in human patients in our study whether or not they were receiving antibiotic therapy. This is in contrast to blood cultures that were positive in only 18% of these same patients, which is similar to levels of detection reported in past clinical studies ( Tsalik et al, 2012 , Gille-Johnson et al, 2013 , Loonen et al, 2014 , Knabl et al, 2016 , Bacconi et al, 2014 ). Indeed, the FcMBL ELLecSA was not only able to detect infection, but to track infection in near real time in our pig infection model regardless of blood culture results.…”

Section: Discussionsupporting

confidence: 85%

“…4 d). In contrast, when we compared these results to blood cultures as a method of direct detection of bacteria, and for which molecular diagnostics have similar detection rates ( Bacconi et al, 2014 , Knabl et al, 2016 ), the blood cultures only detected 18% of infections (12/67) in the sepsis patient cohort ( Fig. 4 d).…”

Section: Resultsmentioning

confidence: 99%

“…Infections that can trigger a systemic inflammatory response syndrome (SIRS) leading to sepsis due to the presence of pathogens or released toxins ( Bone, 1992 ) are most commonly diagnosed using blood cultures in combination with characteristic physiological alterations (e.g., increased heart and respiratory rates, temperature, white blood cells counts) ( Angus and van der Poll, 2013 , Yealy et al, 2014 , Singer et al, 2016 ). But only 15 to 30% of sepsis patients with documented infection produce positive blood cultures, ( Tsalik et al, 2012 , Gille-Johnson et al, 2013 , Loonen et al, 2014 , Knabl et al, 2016 , Bacconi et al, 2014 ) and even then it can often take days before definitive results are obtained. With the absence of direct biomarkers for sepsis, blood proteins involved in the host inflammatory response, such as C-reactive protein (CRP) and procalcitonin (PCT), have been used to detect infection and predict outcome in septic patients ( Pierrakos and Vincent, 2010 , Chan and Gu, 2011 ).…”

Section: Introductionmentioning

confidence: 99%

“…More rapid molecular diagnostic assays (e.g., based on PCR and mass spectrometry) can identify specific types of pathogens, but MALDI mass spectrometry requires a culture step ( Idelevich et al, 2014 ) to expand the number of live pathogens to above 10 5 colony forming units (CFUs), which again limits its use to the minority of patients who are culture positive ( Tsalik et al, 2012 , Gille-Johnson et al, 2013 ). PCR-based methods, such as SeptiFast (Roche, Diagnostics GmbH), hybcell® Pathogens (Anagnostics Bioanalysis GmbH), and IRIDICA (Abbott Molecular) have been developed that can detect specific types of whole pathogens directly in blood within 4–6 h. However, because they require the presence of the pathogen DNA in blood, they fail to detect suspected bloodstream infections in 60–90% of patients ( Knabl et al, 2016 , Bacconi et al, 2014 ). The T2Candida Panel (T2 Biosytems) has demonstrated improved high specificity and sensitivity for fungal infections, and it can identify invasive candidiasis in blood culture negative patients ( Mylonakis et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

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A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood

et al. 2016

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BackgroundBlood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis.MethodsAn Enzyme-Linked Lectin-Sorbent Assay (ELLecSA) that uses magnetic microbeads coated with an engineered version of the human opsonin, Mannose Binding Lectin, containing the Fc immunoglobulin domain linked to its carbohydrate recognition domain (FcMBL) was developed to quantify pathogen-associated molecular patterns (PAMPs) in whole blood. This assay was tested in rats and pigs to explore whether it can detect infections and monitor disease progression, and in prospectively enrolled, emergency room patients with suspected sepsis. These results were also compared with data obtained from non-infected patients with or without traumatic injuries.ResultsThe FcMBL ELLecSA was able to detect PAMPS present on, or released by, 85% of clinical isolates representing 47 of 55 different pathogen species, including the most common causes of sepsis. The PAMP assay rapidly (< 1 h) detected the presence of active infection in animals, even when blood cultures were negative and bacteriocidal antibiotics were administered. In patients with suspected sepsis, the FcMBL ELLecSA detected infection in 55 of 67 patients with high sensitivity (> 81%), specificity (> 89%), and diagnostic accuracy of 0·87. It also distinguished infection from trauma-related inflammation in the same patient cohorts with a higher specificity than the clinical sepsis biomarker, C-reactive Protein.ConclusionThe FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections, even when blood cultures are negative and antibiotic therapy has been initiated. It may help to triage patients with suspected systemic infections, and serve as a companion diagnostic to guide administration of emerging dialysis-like sepsis therapies.

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Section: Discussionsupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood

et al. 2016

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“…Primers: FUNGIf, FUNGIrA, FUNGIrB Probe: FungiProbe challenging technique that can be implemented and performed in a simple laboratory setting. In comparison to other molecular tests, ddPCR assay does not require additional (time-consuming and expensive) DNA enrichment steps prior to DNA extraction, such as centrifugation, filtration or microfluidic steps, to purify pathogens (Wellinghausen et al, 2009;Carrara et al, 2013;Loonen et al, 2014;Stevenson et al, 2016;Knabl et al, 2016;Pfaller et al, 2016;Pilecky et al, 2018). This simplification ultimately results in a rapid identification of pathogens within 4 h (including 1 h hands-on time), whereas most molecular tests require 4-8 h, or longer (Pilecky et al, 2018).…”

Section: Fungi (28s Rrna)mentioning

confidence: 99%

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Wouters

Dalloyaux

Christenhusz

et al. 2019

Microbial Biotechnology

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Summary The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad‐range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10‐fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1–2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52–96) and specificity 87% (95% CI 69–96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.

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DNA Microarray-based Detection of Bacteria in Samples Containing Antibiotics: Effect of Antibiotics on the Performance of Pathogen Detection Assays

Shin

Kim

et al. 2021

Biotechnol Bioproc E

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Evaluation of a multiplex OnSpot Primer-Extension PCR assay in the diagnosis of sepsis

Cited by 7 publications

References 17 publications

A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood

A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

DNA Microarray-based Detection of Bacteria in Samples Containing Antibiotics: Effect of Antibiotics on the Performance of Pathogen Detection Assays

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