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

Abstract

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...

Evaluation of semi-nested polymerase chain reaction (PCR) and mannan antigen detection compared to blood culture for diagnosis of candidemia

“…Thus, this method can be applied as a specific rapid laboratory diagnosis associated with appropriate clinical evaluation to start antifungal therapy in suspected patients (Tirodker et al, 2003;Wellinghausen et al, 2009;Ruppenthal et al, 2005). Similar conclusion was made by a previous study from Egypt with PCR for 16 srRNA (Hassan et al, 2014).…”

Comparison of three different commercial PCR assays for the detection of pathogens in critically ill sepsis patients

“…Combining blood culture and PCR assays could raise the detection rate of microorganisms, as shown by our research and in other recent studies [13,16,20]. Future studies involving a larger number of patients are required to compare different PCR systems and to further evaluate their diagnostic and prognostic validity, as well as the effect the use of these assays has on clinical management and outcome.…”

“…Cultivating organisms from blood cultures-still considered the gold standard for the detection of pathogens-is very time consuming. In a clinical study by Wellinghausen et al [13], the mean time taken to reach a positive result was 19.2 h (range 6.7-58.4). In other cases, up to 120 h have been required [14].…”

“…The other two PCR assays (SepsiTest® and VYOO®) are less well studied in respect to sepsis. In their multicenter study, Wellinghausen et al [13] investigated the SepsiTest® assay using 342 samples from 187 patients with systemic inflammatory response syndrome (SIRS), sepsis or neutropenic fever. They documented a higher rate of positive results as compared to blood cultures.…”

An Evaluation Of Direct Identification Of Pathogens From Blood Cultures By Maldi-Tof Mass Spectrometry

“…But in comparison with other studies our estimated time correspond to with foreign laboratories (10).Some studies even describe MALDI-TOF mass spectrometry advantages compared to other direct methods to identify the bacteria from the blood, for example, using polymerase chain reaction and the latter method is limited by the high cost, not every species of microorganisms is determined in one study, in addition, it is enough difficult to realize in practic (28)(29)(30)(31).…”