Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood

Handschur, Michael; Karlic, Heidrun; Hertel, Christian; Pfeilstöcker, Michael; Haslberger, Alexander G.

doi:10.1016/j.cimid.2007.10.005

Cited by 69 publications

(54 citation statements)

References 34 publications

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“…Our primers can also lead to nonspecific amplification of 150 bp of human chromosome 1, so these cases were classified as negative (data not shown). Some authors have proposed extraction techniques, including a smaller amount of human DNA to eliminate PCR false-positive results, but this can also reduce the amount of bacterial target DNA, thereby leading to reduced sensitivity (25,26). Moreover, to improve performance, it would be interesting to increase the volume of samples analyzed by molecular tools.…”

Section: Discussionmentioning

confidence: 99%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI.

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Section: Discussionmentioning

confidence: 99%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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“…16S rRNA gene-based broad-range PCR is a promising tool for rapid diagnosis of bloodstream infection and several different protocols and systems have been evaluated (9,11,23,25,29,31; for a review, see reference 2). Most studies used a general amplification step, followed by hybridization with probes or microarrays to discriminate between higher taxonomic levels or sequencing of the amplicon.…”

Section: Discussionmentioning

confidence: 99%

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

et al. 2009

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In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 ؉ 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.Bloodstream infection is a life-threatening condition with a high mortality rate, especially in intensive care and neutropenic patients (5,19,35,38). Pathogenic bacteria are the most frequent causes of bloodstream infection, although fungi can also be isolated in a minority of patients (7,17,21,32,34). Currently, inoculation of blood cultures (BC) is the standard method for microbiological diagnosis of bloodstream infections. However, the limitations of BC include relatively low sensitivities and a long time-to-result for detection and identification of the pathogen, generally over 2 days, and even longer for fastidious organisms (13,20,27).In contrast, DNA-based procedures may offer faster and more reliable diagnoses (3, 30). PCR amplification of microbial genes, followed by detection of amplified products by gel electrophoresis or real-time PCR monitoring using fluorescent dyes or target-directed fluorescent probes, is a quick process allowing pathogen detection within a few hours (18). Identification of microorganisms can be performed by PCR algorithms, taxon-specific oligonucleotide microarrays, or sequencing amplicons (30).PCR amplification of conserved regions of the bacterial genome, in particular the 16S rRNA gene, combined with sequence analysis is a well-established technique for the identification of bacterial pathogens (18). The main advantages of targeting the 16S rRNA gene are the broad range of pathogens detectable and the independence of this method from the in vitro viability of strains (6). The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4, 39), have been counteracted by the devel...

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“…However, exogenous DNA contamination during workflow or the presence of DNA traces in PCR reagents, including Taq DNA polymerase from the production strain, may account for false-positive results (53,54). Given these obstacles, diverse approaches have been reported to optimize such techniques and include the use of shrimp nuclease (SNuc) (46), the removal of human background DNA, and bacterial DNA enrichment (55). Despite the attempts to implement sensitive techniques to detect traces of microbial translocation in peripheral blood of HIV-infected patients, concerns remain regarding the risk of contamination.…”

Section: Measurement Of Microbial Translocationmentioning

confidence: 99%

Microbial Translocation in the Pathogenesis of HIV Infection and AIDS

2013

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SUMMARYIn pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections, the translocation of microbial products from the gastrointestinal (GI) tract to portal and systemic circulation has been proposed as a major driver of the chronic immune activation that is associated with disease progression. Consistently, microbial translocation is not present in nonpathogenic SIV infections of natural host species.In vivostudies demonstrated that HIV/SIV-associated microbial translocation results from a series of immunopathological events occurring at the GI mucosa: (i) early and severe mucosal CD4+depletion, (ii) mucosal immune hyperactivation/persistent inflammation; (iii) damage to the integrity of the intestinal epithelium with enterocyte apoptosis and tight junction disruption; and (iv) subverted the gut microbiome, with a predominance of opportunistic bacteria. Directin situevidence of microbial translocation has been provided for SIV-infected rhesus macaques showing translocated microbial products in the intestinal lamina propria and distant sites. While the mechanisms by which microbial translocation causes immune activation remain controversial, a key pathogenic event appears to be innate immunity activation via Toll-like receptors and other pathogen recognition receptors. Accumulating clinical observations suggest that microbial translocation might affect HIV disease progression, response to therapy, and non-AIDS comorbidities. Given its detrimental effect on overall immunity, several interventions to prevent/block microbial translocation are currently under investigation as novel therapeutic agents for HIV/AIDS.

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Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood

Cited by 69 publications

References 34 publications

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

Microbial Translocation in the Pathogenesis of HIV Infection and AIDS

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