Evaluation of a real-time fluorescent PCR assay for rapid detection of Group B Streptococci in neonatal blood

Golden, Stephen M.; Stamilio, David M.; Faux, Brian M.; Cruz, Wilfred P. dela; Shoemaker, Craig T; Blackmon, Camille L.; Stassen, Sarah D.; Clark, Velvet M.; Smith, James W.; Johnson, Oswald L.

doi:10.1016/j.diagmicrobio.2004.04.021

Cited by 23 publications

(10 citation statements)

References 8 publications

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“…27 At Magee Women's Hospital, 70% of our NICU admissions represent near term/term infants who are at risk for systemic infection, whereas the remaining 30% represent preterm infants (data not shown). Standard of care for these infants includes antimicrobial therapy with at least two antibiotics (usually ampicillin and gentamicin in the case of the term infant) for 48 to 72 hours while awaiting culture results.…”

Section: Discussionmentioning

confidence: 89%

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

Jordan

Durso

2005

The Journal of Molecular Diagnostics

146

108

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Speed is of the essence when evaluating an infant with symptoms consistent with sepsis. Because of the high morbidity and mortality associated with neonatal sepsis, infants receive multiple, broad-spectrum antibiotics before receiving finalized blood culture results. Incorporating an additional, reliable, yet rapid assay to detect bacteria directly from blood would facilitate timely diagnosis and appropriate care. To this end, we designed a real-time polymerase chain reaction (PCR) assay targeting the highly conserved 380 bases of 16S rDNA. DNA was extracted from whole-blood samples using a Qiagen column. The limit of detection for the TaqMan-based assay, using a Smartcycler instrument, was 40, 50, or 2000 colony-forming units per milliliter of blood (CFU/ml) of Escherichia coli, group B Streptococcus, and Listeria monocytogenes, respectively, when white blood cell counts were below 39,000/ l. Implementing this approach requires less than 4 hours for both sample preparation and real-time PCR compared with 1 to 2 days to detect growth in culture or 5 days to finalize no-growth culture results. There was an overall agreement between the results of culture and real-time PCR of 94.1% (80 of 85) in this study. These results suggest that molecular techniques can augment culture-based methods for diagnosing neonatal sepsis, especially in infants whose mothers have received intrapartum antibiotic prophylaxis. (J Mol Diagn 2005, 7:575-581)

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Section: Discussionmentioning

confidence: 89%

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

Jordan

Durso

2005

The Journal of Molecular Diagnostics

146

108

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“…assay targeting the CAMP-factor (cfb) gene for the detection of S. agalactiae in neonatal blood (Golden et al 2004), the present assay was more sensitive. Further, low CV values obtained for intra-and inter-runs indicate a high level of assay repeatability and reproducibility.…”

Section: Figurementioning

confidence: 80%

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia

Jiang

et al. 2015

Journal of Fish Diseases

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Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.

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“…Golden et al evaluated a hybridization probe polymerase chain reaction (PCR) assay to detect GBS-specific cfb gene target DNA sequence in blood specimens [49]. Both sensitivity and specificity of the real-time PCR assay were 100%.…”

Section: Early-onset Neonatal Sepsismentioning

confidence: 99%

How Can the Microbiologist Help in Diagnosing Neonatal Sepsis?

Paolucci

Landini

Sambri

2012

International Journal of Pediatrics

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Neonatal sepsis can be classified into two subtypes depending upon whether the onset of symptoms is before 72 hours of life (early-onset neonatal sepsis—EONS) or later (late-onset neonatal sepsis—LONS). These definitions have contributed greatly to diagnosis and treatment by identifying which microorganisms are likely to be responsible for sepsis during these periods and the expected outcomes of infection. This paper focuses on the tools that microbiologist can offer to diagnose and eventually prevent neonatal sepsis. Here, we discuss the advantages and limitation of the blood culture, the actual gold standard for sepsis diagnosis. In addition, we examine the utility of molecular techniques in the diagnosis and management of neonatal sepsis.

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Evaluation of a real-time fluorescent PCR assay for rapid detection of Group B Streptococci in neonatal blood

Cited by 23 publications

References 8 publications

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia

How Can the Microbiologist Help in Diagnosing Neonatal Sepsis?

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