Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

Introduction: Diagnosis of bloodstream infections using bacteriological cultures suffers from low sensitivity and reporting delay. Advanced molecular techniques introduced in many laboratories provide rapid results and may show improvements in patient outcomes. This study aimed to evaluate the usefulness of a molecular technique, broad-range 16S rRNA PCR followed by sequencing for the diagnosis of bloodstream infections, compared to blood culture in different patient groups. Methodology: Conventional PCR was performed, using broad-range 16S rRNA primers, on blood cultures collected from different patients with suspected bloodstream infections; results were compared with those of blood culture. Results: Though blood culture is regarded as the gold standard, PCR evaluation showed sensitivity of 86.25%, specificity of 91.25%, positive predictive value of 76.67%, negative predictive value of 95.22%, and accuracy of 88.8%. Conclusions: Molecular assays seem not to be sufficient to replace microbial cultures in the diagnosis of bloodstream infections, but they can offer a rapid, good negative test to rule out infection due to their high negative predictive value.

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“…These values are similar to those found in other studies using conventional or automated PCR techniques [20,21].…”

Section: Discussionsupporting

confidence: 91%

Evaluation of broad-range 16S rRNA PCR for the diagnosis of bloodstream infections: two years of experience

Hassan

Enany

Rizk

2014

J Infect Dev Ctries

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“…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).…”

Section: Mannan Antigen Detectionsupporting

confidence: 75%

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

Al-Kasaby¹,

Kheir²,

Mefreh³

et al. 2017

Afr. J. Microbiol. Res.

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Invasive Candida infections have emerged as an important pathogen in the last decade, especially in immunocompromized patients. The aim of the present study was to evaluate the detection of Candida species in blood samples from pediatric patients with sepsis by blood culture method versus antigen detection method by enzyme linked immunosorbent assay (ELISA) and molecular method by polymerase chain reaction (PCR). This cross-sectional study was carried out on children at Mansoura University Children Hospital (MUCH) with the presence of clinical signs suggesting sepsis with absence of prior antifungal therapy. Laboratory diagnosis included blood culture, mannan antigen detection of Candida species by ELISA and semi-nested PCR. Candida species were detected by blood culture in 15.6% of the children and was detected by PCR and antigen detection in 20% for each. Candidemia was more frequently detected among patients with central venous lines (38.5%), children with Diabetes Mellitus (DM) (23.1%) and children with frequent blood transfusions (15.4%). However, these risk factors were not statistically significant. Mortality rate among children with candidemia was 42.9%. The sensitivity, specificity, and accuracy of antigen detection method by ELISA compared to culture, were 71.4, 89.5 and 86.7%, respectively. The sensitivity, specificity and accuracy of semi-nested PCR compared to culture were 85.7, 92.1 and 91.1% respectively. It can be concluded from this study that Candida species is a frequent pathogen associated with pediatric sepsis. Blood culture though is a reliable laboratory method for its diagnosis may lack sensitivity and requires prolonged time. Seminested PCR for detection of candidemia is sensitive, specific and accurate method. Mannan antigen detection by ELISA is rapid and easy; however, it may lacks specificity; it can be used as a preliminary method for screening. Further studies are recommended to detect the appropriate laboratory algorithm for early diagnosis of candidemia to start antifungal therapy appropriately.

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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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Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

Cited by 12 publications

References 23 publications

Evaluation of broad-range 16S rRNA PCR for the diagnosis of bloodstream infections: two years of experience

Evaluation of broad-range 16S rRNA PCR for the diagnosis of bloodstream infections: two years of experience

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

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

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