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A new twist on the detection of disease-causing microbes.

Most infectious disease in vitro diagnostic (IVD) products fit into a limited number of broad categories. Direct culture, although not widely used, is one means of detecting an infectious agent in a clinical specimen. There are those tests that are immunoassay-based and rely upon the measurement of the patient's antibody to an infectious agent as a means of documenting or confirming exposure to that agent. Some immunoassays utilize capture-and-detection antibodies or other complementary biomolecules to directly measure the presence of an infectious agent or a byproduct of that agent. Finally, there are nucleic acid-based molecular assays that measure amplified genomic DNA (or RNA) from the infectious agent. All of these are useful tools to help identify disease-causing pathogens and serve as useful aids in the diagnosis of infectious diseases. However, all have their shortcomings.

Antibody detection assays rely upon the immune response and therefore are an indirect means of determining exposure to an infectious agent. Also, cross-reacting antibodies or non-specific interactions can potentially lead to false results. Antigen-capture immunoassays are extremely reliant upon the avidity and specificity of the antibodies employed. They too can be under- or oversensitive, depending on how they are configured and the quality of the antibodies. Genomic PCR tests can be very sensitive and specific, but the presence of genomic DNA does not necessarily correlate with the presence of live, viable microbes, as target DNA may have come from non-viable organisms in the blood or cell-free microbial DNA from contamination or remote infections sites. (1)

There is a new technology that is capable of detecting any viable microbe in virtually any specimen in approximately two hours rather than days. This technology utilizes, in part, the microbe's own machinery to generate the assay result and has been termed Enzymatic Template Generation and Amplification (ETGA). ETGA relies upon the fact that all viable microbes possess DNA polymerase. It is the microbe's endogenous DNA polymerase that is the key component of the ETGA technology.

How ETGA works

The technology has been explained in detail elsewhere; (2) please review the referenced article in the journal Nucleic Acids Research. Simplified, the ETGA assay is configured with a very specific oligonucleotide substrate (Figure 1). The substrate consists of two relatively short oligonucleotides annealed together, yielding a nucleic acid substrate that is capable of being extended by endogenous microbial DNA polymerase. Substrate modified by DNA polymerase serves as a target for a quantitative polymerase chain reaction (qPCR). If there is no microbial DNA polymerase, there is no substrate modification and therefore a negative qPCR outcome. If there are viable microbes present, their DNA polymerase extends the substrate and a positive qPCR is detected.

The typical ETGA assay involves initial steps designed to isolate any potential microbes from the specimen in question. This "sample" is then lysed in the presence of the ETGA reaction components and then incubated for a brief period to allow DNA extension to occur if DNA polymerase is present. An aliquot of this material is then combined with the qPCR master mix and amplified via typical thermocycling protocols.

It is important to note that while any microorganism will yield a positive result in the ETGA reaction, the assay will not identify the type of organism that produced the positive result. Studies have shown, however, that ETGA sample preparation results in ample amounts of the organism's genomic DNA. Positive ETGA specimens could be subsequently analyzed by any form of genomic DNA analysis. (3) It is equally important to emphasize that the ETGA reaction requires viable microbes. Only those organisms with active, functional DNA polymerase will yield a positive qPCR following the ETGA assay.

Potential applications of the ETGA assay

The ETGA assay is ideally suited for analysis of any normally sterile fluid. By definition, a sterile fluid should be free of any viable microorganism and therefore should be free of functional DNA polymerase.

Extensive feasibility and clinical studies have been done using the ETGA assay. (3), (4) One of the most investigated applications is the utilization of ETGA as a means to expedite traditional blood culture (BC) results. Bacteremia, fungemia, and septicemia contribute to significant morbidity and mortality. There are more than 200,000 deaths in the U.S. per year due to systemic microbial infection of the bloodstream. (5) BC is the gold standard for evaluation of those patients suspected of bloodstream infections (BSI), and the standard BC typically takes from one to five days before results can be interpreted. During that period, physicians frequently administer broad-spectrum antibiotics empirically, which can contribute to widespread overuse of antibiotics. ETGA studies have demonstrated that positive BC results can be shortened by threefold compared to their normal flip time. (3) The modeling studies suggest that a negative BC might be determined by ETGA in as little as one to two days, as opposed to five days for traditional BC.

In the ETGA BC assay, following inoculation and a brief incubation period, a sample is drawn from the BC bottle. Using a differential lysis procedure and centrifugation steps, potential microbes are separated from human blood cells. The ETGA assay is carried out as described above, and the qPCR amplification curve is interpreted versus a normal background threshold that was previously established using many microbe negative BC bottles as controls. These studies have demonstrated that ETGA is capable of detecting as few as ten colony forming units (CFU)/mL of gram positive or gram negative bacteria. (2)

Another well-studied potential application of the ETGA assay is for sterility testing of platelet concentrates (PC). More than 1.5 million units of platelet products are transfused in the United States each year. (6) Before PC can be transfused into a patient, they must undergo extensive sterility testing to verify that there is no microbial contamination of the PC. Transfusion of contaminated PC into a patient can lead to serious health implications and even death.

Currently, BC is the gold standard for PC sterility testing. PC are only suitable for transfusion for a period of five days after collection, and typically two of the five days are consumed while awaiting the results of the BC sterility test. ETGA PC studies have shown that this sterility testing can be shortened significantly since positive results were detected, on average, threefold sooner with ETGA-PC assay as opposed to BC. More studies are warranted; however, such testing could significantly extend the period of time PC are available for transfusion prior to expiration.

Since ETGA is universal in its microbial detection capabilities and has been demonstrated to be so sensitive in certain modeling studies (sensitivity experiments indicate that a single microbe could be detected with this assay) (2), there are numerous possible applications for the ETGA assay. Some other theoretical applications ideally suited for ETGA analysis might be:

1. Rapid anti-microbial susceptibility testing. (4) Experiments have shown that ETGA can be used to quickly assess whether or not microbes are susceptible to certain antibiotics. This results in a rapid phenotypic analysis, and not a genotypic analysis.

2. Rapid microbiological methods. ETGA could be used for bioburden analysis of finished products or in-process materials in many industries, including the pharmaceutical industry.

3. Assessment of normally sterile body fluids such as cerebral spinal fluid.

4. Looking for the presence of, or to quantify the number of, viable microbes in certain foods or beverages.

5. Identifying the presence of, or quantifying the amount of, viable microbes in certain environmental specimens such as water.

The ETGA technology provides for a novel way of analyzing specimens for viable microbes. As a diagnostic tool or as a microbiological tool, there are likely many more applications for an assay that is so sensitive yet is so universal.

References

(1.) Ecker DJ, Sampath R, Li H, et al. New technology for rapid molecular diagnosis of bloodstream infections. Expert Rev Mol Diagn. 2009;10(4):399-415.

(2.) Zweitzig DR, Riccardello NM, Sodowich BI, O'Hara SM. Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes. Nucl Acids Res. 2012;40(14):e109.

(3.) Zweitzig DR, Sodowich BI, Riccardello NM, O'Hara SM. Feasibility of a novel approach for rapid detection of simulated bloodstream infections via enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA polymerase activity. J Mol Diagn. 2013;15(4):319-330.

(4.) Sodowich BI, Zweitzig DR, Riccardello NM, O'Hara SM. Feasibility study demonstrating that enzymatic template generation and amplification can be employed as a novel method for molecular antimicrobial susceptibility testing. BMC Microbiol. 2013;13(1):e.191.

(5.) Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med. 2001;29(7):1303-1310.

(6.) Stroncek DF, Rebulla P. Platelet transfusions. Lancet. 2007;370(9585):427-438.

Mark Kopnitsky is Vice President of Science and Quality at ZEUS Scientific.
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Title Annotation:Special feature
Author:Kopnitsky, Mark J.
Publication:Medical Laboratory Observer
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2013
Words:1477
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