Evaluation of an automated microbiologic blood culture device for detection of bacteria in platelet components

Wagner, Stephen J.; Robinette, Daniel

doi:10.1046/j.1537-2995.1998.38798346637.x

Cited by 78 publications

(83 citation statements)

References 26 publications

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“…As a result, false-negative results caused by sampling errors are a problem for both microbiological and molecular genetic methods. Microbiological detection using 10 to 20 ml of platelet concentrate for aerobic and anaerobic cultivation is susceptible to false-positive results (37) and requires a long incubation time, while NAT assays should be sensitive and fast enough for a routine contamination screening of PCs. Therefore, we suggest the screening of PCs during the second day after donation, for which a satellite bag of the PCs should be used.…”

Section: Discussionmentioning

confidence: 99%

“…The detection of bacterial growth by culture methods is still the most sensitive method. A bacterial sterility test should be rapid, affordable, adequately sensitive, specific, and simple to perform (37). Only automated bacterial blood culturing systems meet many of the requirements of an ideal test because they detect a wide range of organisms at concentrations of only 1 to 10 CFU per ml (5).…”

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confidence: 99%

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Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

2004

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The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

2004

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“…Culture methods, which require a long time to demonstrate the presence of bacteria, have remained the preferred way for platelet bacteria screening, although bacteria in PCs have been detected with other approaches (18 ). A bacterial-detection test should be rapid, affordable, adequately sensitive, specific, and simple to perform (19,20 ). Although no single approach now meets all these criteria, molecular biological approaches offer promising opportunities for detecting contaminating organisms in blood components.…”

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confidence: 99%

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Störmer

Kleesiek

Dreier³

2007

Clinical Chemistry

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Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid-binding matrices. We adapted this technology with a broadrange 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresisderived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primerprobe system and locked nucleic acid technology. Coamplification of human ␤ 2 -microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead-based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 ؋ 10 3 colony-forming units (CFU)/L for S. epidermidis and 22 ؋ 10 3 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid

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“…The screening methods used by different centers are visual inspection, microscopic examination of stained smears, pH and glucose dipstick tests (29), and tests with automated culture systems (7,28). No screening method, however, satisfies the main requirements of high sensitivity, high specificity, and rapidity.…”

Section: Discussionmentioning

confidence: 99%

Rapid Screening Method for Detection of Bacteria in Platelet Concentrates

Ribault¹,

Harper²,

Grave³

et al. 2004

J Clin Microbiol

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Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 10 2 CFU/ml, depending upon the bacterial strain.

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Evaluation of an automated microbiologic blood culture device for detection of bacteria in platelet components

Abstract: Data from this preliminary evaluation suggest that sampling times of 24 hours or more would be necessary to provide confidence in detection of E. coli or S. epidermidis in PCs using this culture method.

Cited by 78 publications

References 26 publications

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Rapid Screening Method for Detection of Bacteria in Platelet Concentrates

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