Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: a case control study

Reier‐Nilsen, Tonje; Farstad, T; Nakstad, Britt; Lauvrak, Vigdis; Steinbakk, Martin

doi:10.1186/1471-2431-9-5

Cited by 97 publications

(98 citation statements)

References 30 publications

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“…The diagnostic performances of these in-house assays varied considerably (for a review, see reference 18). For instance, in a study with 48 newborns admitted to the ICU with suspected sepsis, the PCR detection assay with sequencing of a limited number of samples revealed a diagnostic sensitivity of 66.7% and a specificity of 87.5% (26). In another study, PCR and hybridization probes differentiating between gram-positive and gram-negative taxons to detect bacteremia in 548 blood samples from newborns (14) resulted in a sensitivity and specificity of 96 and 99.4%, respectively.…”

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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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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“…were the most frequently detected. In a cohort of newborns with sepsis, other authors evaluated amplification of the bacterial 16S rRNA gene for the early diagnosis of bloodstream infection: compared to blood culture, the sensitivity was 66.7% and the specificity was 87.5% (19). These percentages are markedly lower than the 85% and 93.5%, respectively, that we observed in the present study for the SeptiFast test compared to blood culture, reinforcing the evidence of its higher performance than previous PCR-based methods.…”

Section: Discussionmentioning

confidence: 99%

Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis

et al. 2011

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Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] ‫؍‬ 78.7 to 89.7%) and a specificity of 93.5% (95% CI ‫؍‬ 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n ‫؍‬ 97 [5.8%] versus n ‫؍‬ 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.Pediatric patients with severe trauma and burns, immunodeficiency, malignancy, and prematurity have an increased incidence of septicemia with a high case fatality rate (10 to 50%) (10). Moreover, prolonged hospitalization, broad-spectrum empirical antimicrobial therapy, and supportive care have a strong impact on the cost of care (15,25).Oncohematological patients and newborns, particularly preterm infants, are at high risk for severe infections and sepsis due to their deficient and/or immature immunologic defense (5, 7). Rapid detection of the infectious cause and prompt initiation of appropriate antimicrobial treatment are fundamental for the successful treatment of septic patients and for the reduction of antibiotic resistance rates (23, 30).Blood culture is the current "gold standard" for the detection of bloodstream microbial pathogens; although it allows microbes to be identified and their susceptibility profiles to be tested, it presents several limitations. Lack of rapidity is a major problem: detection of bacterial growth requires approximately 12 to 48 h or more in the case of fastidious bacterial or invasive fungal infection (1, 18). Another remarka...

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“…In order to estimate the true concentration of pathogen DNA available in whole blood for molecular analysis from patients with bloodstream infections, we analyzed the data available in the literature. Organism-specific quantitative PCR has been used to measure the bacterial DNA present in whole-blood specimens from patients with sepsis, pneumonia, or suspected bloodstream infections (13,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). Each of these studies analyzed whole-blood specimens from patients with suspected or confirmed infections rather than spiked samples for which the genome-to-viable cell ratio is expected to be close to 1:1.…”

Section: Repeated Analytical Testing By Pcr/esi-msmentioning

confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

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Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: a case control study

Cited by 97 publications

References 30 publications

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

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

Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

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