Molecular Diagnosis of Bacteremia

a bacteremia and molecular diagnostic technology, applied in the field of clinical diagnostics, can solve the problems of inability to quickly and accurately identify patients with bacterial infections, potential delays in diagnosis and treatment, and increased risk of morbidity and mortality, and achieve the effect of sensitive and accurate detection of bacterial microorganisms

Inactive Publication Date: 2009-08-27
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]These and other embodiments of the invention provide the art with methods and tools for sensitively and accurately detecting bacterial microbes in a variety of sample types.

Problems solved by technology

There are an estimated 500,000 patients in the United States who develop bacteremia, with mortality rates ranging from 25-50%.1 Early recognition and aggressive therapeutic intervention is known to significantly improve outcomes for those with systemic bacterial infections.1 Unfortunately, no definitive clinical parameters or diagnostic assays currently exist that allow clinicians to rapidly and accurately identify patients with bacterial infections among those in whom systemic infections are suspected.
Early systemic bacterial infections, or those which occur in vulnerable or immunosuppressed hosts may be more subtle however, leading to potential delays in diagnosis and treatment with associated increased risk for morbidity and mortality.
Further, inherent limitations of the ‘gold standard’ diagnostic test for bacteremia, blood culture, renders it ineffective for guiding acute management decisions.
These limitations include significant time delays associated with reporting of positive findings (typically at least 24-48 hours), relatively low sensitivity ranging from 30-50% among patients who meet criteria for sepsis syndrome, and diminished sensitivity in patients already on antibiotics.
The failure of either clinical judgment or diagnostic technology to provide quick and accurate data for identifying patients with bacteremia, leads most clinicians to follow a conservative management approach for those in whom systemic infection is suspected.
Well known clinical examples in which patients are routinely hospitalized and given antibiotics while awaiting blood culture results include febrile episodes in infants, due to the high mortality associated with unrecognized septicemia, and any febrile illness in intravenous drug users due to the high risk of life-threatening infective endocarditis which is principally characterized by the presence of bacteremia.
The benefits of conservative management may be offset however, by added costs and potential iatrogenic complications associated with treatment and hospital days for those later found not to be bacteremic, as well as increased rates of antimicrobial resistance.
Findings published to date have principally been restricted to detection of bacteria in highly infected tissue specimens, e.g. resected heart valves in patients with suspected infective endocarditis, or clinical samples from an infected site such as an abscess.
Unfortunately, less success has occurred with universal screening of blood samples, principally due to technical problems of the assay, most commonly related to contaminant bacterial DNA.

Method used

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  • Molecular Diagnosis of Bacteremia
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Study Design

[0029]This was a prospective identity-unlinked investigation. This technique, is a sampling method which allows single point in time patient related data collection with anonymous testing of patient blood samples taken for various blood borne pathogens. In brief, excess serum was retained from patients' 18 years of age and older who presented to The Johns Hopkins Hospital Emergency Department and had blood drawn for blood culture. Enrolled patients were assigned a unique study number, which was used to code the excess sera, as well as the laboratory and descriptive data. Descriptive data included demographics, clinical data, discharge diagnosis, and blood culture findings. After coding, all patient identifiers were stripped. In this way, results of the PCR analysis could not be directly linked to a patient by name or history number. The study was approved by the Johns Hopkins University Institutional Review Board.

Patients and Sample Collection

[0030]Practice at our hospit...

example 2

[0041]This example demonstrates the contamination present in PCR reagents and the efficacy of the subject method for destroying the contaminants.

[0042]The PCR amplification assay was first tested in a mock sample containing water, with no added bacteria to determine whether the reagents themselves contained contaminating sources of bacterial DNA that might lead to false-positive results. FIG. 2 shows results of a PCR amplification using the 16S rRNA primers described in FIG. 1. Lane 1 shows a product of the expected size, indicating that contaminant bacterial DNA is being amplified in our system. The effect of a predigestion step, in which AluI at is added to all components of the PCR cocktail is shown in Lane 2. (Titration of AluI from 1-20 U / reaction identified 10 U / reaction to be the optimal enzyme concentration.) Lane 3 shows results of a PCR amplification carried out using whole blood taken from a healthy control, with no bacteria added, again showing the absence of a signal. L...

example 3

[0043]This example demonstrates the successful practice of the method using known bacteria spiked into test samples.

[0044]Healthy human whole blood was next spiked with 1 of 4 bacterial isolates and DNA was then extracted. A series of PCR amplifications reactions were subsequently carried out with the decontamination step, described above included, i.e., all PCR reagents were pretreated with the restriction enzyme, Alu I at 10 U / reaction prior to amplification. FIG. 3 shows the PCR amplified products from the whole blood specimens, as well as the negative control samples, in which no bacteria was added prior to PCR amplification. The amplified product of 411 base pairs was detected for each of the spiked reactions. DNA sequencing correctly confirmed the identity of each spiked pathogen.

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Abstract

A highly specific assay can be used for the detection of bacteremia in the clinical setting. The ubiquitous background endogenous DNA present in all PCR reagents is eliminated using a restriction endonuclease digestion. Universal primers for eubacteria are used for detection, and specific primers or probes for bacterial species can be used for identification of species.

Description

[0001]This application claims priority to provisional U.S. Application Ser. No. 60 / 229,376, filed Aug. 31, 2000, the disclosure of which is expressly incorporated herein.FIELD OF THE INVENTION[0002]The invention relates to the field of clinical diagnostics. In particular, it relates to the field of detection of bacteremia in patients.BACKGROUND OF THE INVENTION[0003]There are an estimated 500,000 patients in the United States who develop bacteremia, with mortality rates ranging from 25-50%.1 Early recognition and aggressive therapeutic intervention is known to significantly improve outcomes for those with systemic bacterial infections.1 Unfortunately, no definitive clinical parameters or diagnostic assays currently exist that allow clinicians to rapidly and accurately identify patients with bacterial infections among those in whom systemic infections are suspected.[0004]Patients with fulminant bacteremia are usually easily recognized by the presence of fever and significant vital si...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12P19/34
CPCC07H21/04C12Q1/689C12Q1/6806
Inventor ROTHMAN, RICHARD E.MAJMUDAR, MAULIK D.
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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