Blood Infection Diagnosis by 16S rDNA Broad-Spectrum Polymerase Chain Reaction: The Relationship Between Antibiotic Treatment and Bacterial DNA Load

Sakka, Samir G.; Kochem, Anna-Julia; Disqué, Claudia; Wellinghausen, Nele

doi:10.1213/ane.0b013e3181b79904

Cited by 11 publications

(9 citation statements)

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“…Prior antimicrobial therapy probably contributed to the failure to recover organisms in culture that were detected by PCR, though it is important to recognize that the PCR procedure used in this study was designed to detect only viable microbes. This perspective is supported by conclusions from previously published studies of sepsis in which PCR-positive specimens remained culture negative, presumably due to prior antimicrobial therapy (17,23). In some patients, only a single blood culture specimen was collected, so it is possible that inadequate sampling may have contributed to false-negative cultures, though patients with IE are generally persistently bacteremic (22).…”

Section: Resultsmentioning

confidence: 66%

Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis

et al. 2011

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Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.

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Section: Resultsmentioning

confidence: 66%

Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis

et al. 2011

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“…A smartly designed approach is based on a selective first-step lysis of human blood cells by chaotropic buffers and quantitative degradation of human nucleic acids via chaotroph-resistant DNases (MolYsis, Molzym, Bremen, Germany; [82,83]). The enzyme is subsequently heat-inactivated, and bacterial pathogen cells are enzymatically lyzed in a second step to gain their genomic DNA for NAT detection purposes.…”

Section: Cell Lysismentioning

confidence: 99%

Nucleic Acid Amplification Techniques

Lehmann¹,

Schmitz

2015

Modern Techniques for Pathogen Detection

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Nucleic acid amplification techniques (NATs), more commonly but imprecisely called polymerase chain reaction (PCR), have evolved, among others, as central tools for pathogen identification and strain typing. The huge field of applications of the technique in all its modifications already marks the centerpiece of diagnostic approaches and has not only opened the door to a completely new understanding of elementary and sophisticated metabolic processes but also is the basis of the most important trends in life science industries. In a sense, it acts as the lens of a molecular microscope which visualizes the code from the genomic book, while its meanings have to be deciphered by a further armory of laboratory avenues.In 1983, PCR was invented by Kary Mullis, for which he later (1993) was awarded with the Nobel Prize in Chemistry along with Michael Smith [1]. The protocol is based on targeting specific DNA or rDNA sequences with more or less specific short tracer sequences (primers/oligonucleotides, colloquially oligos) binding to flanking regions of the core sequence to be proliferated. A succession of thermal incubation steps defined by length and temperature are cycled and conduct double-stranded DNA (dsDNA) melting, that is, separation of the target DNA into antiparallel, complementary, single-stranded DNA (ssDNA) strands, specific binding of primers (annealing), and enzymatic amplification out of the basic set of four nucleotide monomers of the sequence embedded. In continuation, the newly synthesized dsDNA copies, selected in length by the specific primer binding sites (PBSs), themselves serve as templates for further replication rounds, setting in motion a chain reaction in which the DNA template is exponentially amplified. The core enzyme is a DNA polymerase, which was heat-sensitive at time of invention but subsequently was destroyed at the melting temperature. To gain useful numbers of target copies, an expensive and time-consuming replacement of the enzyme was needed at the beginning of each of the usually 20-40 replication cycles. The discovery of an extraordinarily thermostable DNA polymerase purified from the thermophilic bacterium Thermophilus aquaticus, which naturally lives in hot (50-80 ∘ C) environments such as hot springs [2], Modern Techniques for Pathogen Detection, First Edition. Edited by Jürgen Popp and Michael Bauer.

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“…Among them, panbacterial and panfungal rRNA gene PCR followed by sequencing identifies the broadest range of pathogens (7)(8)(9). Here, we evaluated the usefulness of a broad-range PCR test, SepsiTest (6,(10)(11)(12)(13)(14), for the monitoring of patients for DNA of pathogens in the blood during ECMO.…”

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confidence: 99%

“…Duplicate samples (1 ml) of EDTA blood (from the same venipuncture as blood culture [BC]) were analyzed using SepsiTest (Molzym, Bremen, Germany) (6,(10)(11)(12)(13)(14), which supplies protocols and reagents for DNA extraction, 16S and 18S rRNA gene PCR, and negative, positive, and internal PCR controls. Amplicon sequencing was done by an overnight service.…”

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confidence: 99%

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Monitoring of Patients Supported by Extracorporeal Membrane Oxygenation for Systemic Infections by Broad-Range rRNA Gene PCR Amplification and Sequence Analysis

Orszag

Disqué²,

Keim³

et al. 2014

J Clin Microbiol

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eThe rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative. P atients supported by extracorporeal membrane oxygenation (ECMO) are at high risk for microbial systemic infections (1, 2) and therefore are continuously monitored by clinical and inflammatory markers. Microbiological results are used to tailor antibiotic treatment to the most effective regimen. Unfortunately, etiologies are typically identified within 1 to 2 days, and up to 31% of cultures remain negative because of limited blood volume analyzed, growth failure of fastidious organisms, inhibition of growth of pathogens by antibiotics, and other unknown reasons (3, 4). Molecular methods are discussed as tools for improvement of the diagnosis of septicemia (5, 6). Among them, panbacterial and panfungal rRNA gene PCR followed by sequencing identifies the broadest range of pathogens (7-9). Here, we evaluated the usefulness of a broad-range PCR test, SepsiTest (6, 10-14), for the monitoring of patients for DNA of pathogens in the blood during ECMO.The study was approved by the Ethics Committee of Hannover Medical High School. SepsiTest results were not considered for the administration of antibiotics. ECMO criteria included cardiac or respiratory failure with beginning or preexisting organ failure.Single pairs of aerobic/anaerobic Bactec Plus bottles (BD, Heidelberg, Germany) and Bactec Mycosis-IC/F bottles were incubated each with 10 ml of blood collected from a venipuncture. Incubation was for 7 (bacteria) or 14 (fungi) days in case of negative results. Species were identified using Vitek 2 (bioMérieux, Marcy l'Etoile, France). Other samples were analyzed according to standard microbiological operations of the laboratory.Duplicate samples (1 ml) of EDTA blood (from the same venipuncture as blood culture [BC]) were analyzed using SepsiTest (Molzym, Bremen, Germany) (6, 10-14), which supplies protocols and reagents for DNA extraction, 16S and 18S rRNA gene PCR, and negative, positive, and internal PCR controls. Amplicon sequencing was done by an overnight service. BLAST results (www.sepsitest-blast.net) were classified at the species (Ն99% sequence identity) and genus (Ն97%) levels (12). Corynebacteria, viridans streptococci, coagulase-negative staphylococci (CNS), and Klebsiella oxytoca/Enterobacter cloacae were not resolvable at the species level. Mixed sequences were resolved by Ripseq analysis (Isentio, Paradis, Norway).Defined criteria were applied to interpret BC-negative, SepsiTest-positive results. "Probable" systemic infection was supposed if (i) results were supported by cultured material (12) before, during, or after ECMO and/or (ii) organisms were found in repeated blood draws within 48 h that were regarded clinically significant on the grounds of the c...

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Blood Infection Diagnosis by 16S rDNA Broad-Spectrum Polymerase Chain Reaction: The Relationship Between Antibiotic Treatment and Bacterial DNA Load

Cited by 11 publications

References 3 publications

Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis

Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis

Nucleic Acid Amplification Techniques

Monitoring of Patients Supported by Extracorporeal Membrane Oxygenation for Systemic Infections by Broad-Range rRNA Gene PCR Amplification and Sequence Analysis

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