Special Feature

How rapid is rapid?

The hidden wait times in PCR

By Jon Faro, MD, PhD

A thin BioNanoPore membrane is shown being placed over a trypticase soy agar (TSA) plate, after first culturing for five hours. Micro-colonies of Staphylococcus aureus were visible after ten minutes of staining.

There is no shortage of DNA sequencing technologies on the market or in development today. These technolgies aim to overcome the long wait times associated with culture-based tests, while striving to offer the same sensitive and specific results found in conventional microbe detection. Many think they are the ultimate solution for diagnosing and treating microbial infections more quickly and with more targeted antibiotics and they do hold promise. But rapid tests have shortcomings—in complexity, in expense, and in some instances in 18-hour overnight enrichment periods—that have left the healthcare community wanting more.

These hurdles have prompted the U.S. Food and Drug Administration (FDA) and the Infectious Diseases Society of America to call for greater accessibility and speed in rapid tests. At a 2009 workshop, these two organizations recommended the development of microbial diagnostics that could be conducted 24/7, were available within a few hours, were of minimal complexity and affordable price, and that could demonstrate reduced antibiotic use and improved patient outcomes.¹ After more than a decade of the medical community wishing for molecular diagnostics to be more than it is, perhaps it is time to look toward more novel microbial approaches to achieve the simple, cost-effective, fast results everyone is searching for.

The complications of waiting

Today there are two popular options for definitive microbial diagnostics: traditional cell culture and polymerase chain reaction (PCR). Depending on the bacteria, cell culture can take anywhere from 24 hours to 21 days. Yet, it is a tried and true method that many medical facilities depend on.

PCR is faster but does not always offer the best solution in detecting microbes quickly. There are a number of reasons for this.

• Though the actual detection process may only take a few minutes, PCR often requires lengthy preparation and overnight enrichment of the assays. Additionally, because of the expense of PCR tests, labs often wait to collect a "critical mass" of samples before running a test. Though results are available within five minutes, preparation of the sample may take as long as 18 hours before it can be tested in the machine to detect the presence of microbial DNA. When this extra time is taken into consideration, more than 24 hours may ultimately be required before PCR can yield results.
• The preparation of a PCR sample is time consuming. The sample must be laboriously processed first before it can be used, and it requires considerable effort. It must not be contaminated with other DNA from live or dead microbes, or else the result will be falsely positive. Separate rooms are often required for DNA extraction and the dedicated machine, in order to prevent cross-contamination.
• PCR requires fresh samples in order to reduce the likelihood of contamination. But for transport, samples must be frozen using a freezing agent (e.g., liquid nitrogen) and kept in that state (using Dewar flasks), which are not available in many settings, especially in developing regions or on the periphery of the clinical setting.
• It is sometimes necessary to "quash" or break cells in the sample so DNA can be readily identified. This process requires fine manual dexterity from the technician. It can also be a problem for thick-walled cells, such as tuberculosis. If this procedure is not done, or done incorrectly, it may result in false negatives.
• PCR requires special equipment and reagents, and this can be a major hurdle to its mass adoption. It requires several primers and reagents and repeated micropipetting in each use. Fine glassware and expensive reagents like Taq DNA polymerase require careful storage and handling to avoid contamination.
• Such complexity and specialized equipment means that only highly skilled laboratorians should use PCR to ensure consistent results. Training is required for proper handling and processing of samples, as well as handling the machine. Training is also necessary to ensure that PCR operators safely handle toxic reagents and biohazardous samples. Laboratorians also must know how to purify DNA samples from contaminants and errant DNAs, a process that often involves hazardous chemicals such as chloroform and acid phenols. And, results are considered inaccurate if proper steps are not followed, requiring another round of time-consuming sample preparation.

None of this is to say that the PCR emperor is wearing no clothes. But it is to suggest that the monarch may not have quite the fashion sense that PCR's advocates claim for him.

Today's truly rapid tests

All of its expense and process complexity makes PCR impractical to use in rural or developing settings, where the fight against microbes and infectious diseases is perhaps most grimly waged. In more developed regions and hospitals, the battle is particularly focused on hospital acquired infections (HAIs) and "superbugs" such as methacyllin-resistant s. aureus. (MRSA). In both settings, lab technicians are under pressure to conduct tests faster, with simpler methods that can be performed close to the patient. As such, demand is growing for fast, easy-to-use and accurately sensitive diagnostic tests to address current infectious disease trends. This has the clinical community looking outside of PCR for solutions.

Several biotechnology innovations have emerged recently to yield microbial detection as fast as the PCR, but without the associated lengthy and laborious sample preparation and wait times.

A simple first step is to determine if bacteria are present in a food or drinking source. The hours of waiting required by culture and expense involved in PCR make these techniques ill-suited for a question which requires an immediate answer. Maryland-based New Horizons Diagnostics has developed an adenosine triphosphate (ATP)-based assay which allows for the detection of a bacterial or fungal contaminant in as little as five minutes. For this assay, a sample contaminated with a bacterial pathogen is treated with a reagent which lyses the cells, liberating ATP. Luciferase is added, and any ATP present will result in a release of energy which may be directly measured.

An entirely different approach is to examine the great metabolic complexity of these pathogens. California-based Biolog has taken the approach of characterizing the bacterial phenotype, using traits such as utilization of metabolic substrates, pH, and oxidation/reduction potential to provide a "metabolic fingerprint," or phenotype microarray. Samples are processed on a 96-well plate, and as many as 50 plates may be run at one time. Results are provided in two hours, plus additional prep time. While this technique relies on an additional culture step before a pathogen may be isolated, it allows for the identification of more than 2,600 microbial species.

While both of these methodologies provide unique approaches to the detection of microbial pathogens, the primary objective is to incorporate speed and specificity. The Ohio biotech firm NanoLogix offers an approach that does just that. It has developed a unique advanced-culture method for detecting and identifying micro colonies two to 24 times faster than traditional Petri cultures. Test times for cholera, bubonic plague, anthrax, and group B streptococcus all come in less than one hour total, compared to 18 to 72 or more hours. Other bacteria, such as MRSA, return results in six hours. E.coli returns in four hours, compared to the 18 to 24 of PCR and petri. Finally, tuberculosis, which is very hard to test using PCR and takes 21 days with cultures, can be diagnosed with NanoLogix technology in just four days.

The technology is fast, accurate, and cost-effective, using the same principles of traditional Petri culture. It does not speed up cell growth; it simply allows technicians to see the growth sooner. Plus, the simple design enables quick integration in the laboratory that can be used after simple training. Recently, NanoLogix published a description of the technology as it relates specifically to group B streptococcus in the American Journal of Perinatology.²

Another novel approach for the rapid detection of bacterial pathogens is coming out of the University of Georgia. There, researchers in the College Of Veterinary Medicine recently published a paper describing a new method of detecting influenza virus rapidly using gold nanoparticles.³ The technology is based on the fact that nanoparticles have different traits according to their size. The method involves coating the gold particles with selected antibodies, which will bind to specific strains of flu viruses. By measuring the laser light particles reflected by gold, researchers can detect influenza viruses within minutes at the cost of only a fraction of a penny per exam.

Where new directions may lead

The consequence of these truly rapid test times is that physicians can make more accurate diagnoses in shorter amounts of time and perhaps refrain from administering broad spectrum antibiotics until the exact bacteria causing the illness is known. This could lead to a vast reduction in the overuse of antibiotics and thus reduce antibiotic resistance.

As the demand for fast, simple, and cost-effective diagnostics grows, the medical community will continue to innovate towards the goal. Over the next few years, it can be expected that more novel rapid approaches will surface and lead towards the more accurate, rapid, and efficient diagnostics that more effectively equip healthcare providers to fight infectious diseases.

Serological and genetic tests are an integral component of CD diagnostics and diet monitoring. They not only facilitate the diagnosis of symptomatic CD, sparing many patients the discomfort and stress of a biopsy, but they also enable cases of asymptomatic, atypical, silent, and latent CD to be more easily recognized.

In pediatrics, all cases of unexplained growth disorders, retarded development, and chronic diarrhea should be considered for the possibility of CD. Likewise, patients with autoimmune diseases such as type 1 diabetes mellitus, certain chromosomal aberration diseases, osteoporosis, pregnancy problems, or lymphoma, which are known to be associated with CD, should also be screened. The importance of identifying individuals with non-classical forms of the disease is underscored by the risk of long-term health consequences such as the development of tumors. Further studies will help to establish the exact burden of CD on the population.

Figure 1. EmA detected by indirect immunofluorescence

Figure 2. Anti-tTG ELISA

Figure 3. Gliadin-analagous fusion peptide (GAF-3X)

Figure 4. EUROArray procedure

Figure 5. Microarray evaluation

At the pilot’s start in January 2009, the hospital’s urine contamination rate exceeded 20 percent. After implementing standardized Lean Sigma processes, it fell to less than six percent in just 11 months. It declined to nearly one percent by September 2010 as these standardized processes gained greater acceptance.

CAPTION

Reference

1. ISDA Public Policy: An unmet medical need: rapid molecular diagnostics tests for respiratory tract infections. Clin Infect Dis. 2011;52 (suppl 4);S384-S395.

2. Faro J, Katz A, Bishop K, Riddle G, Faro S. Rapid diagnostic test for identifying group B streptococcus. Am J Perinatol. 2011;28:(10):811-814.

3. Driskell JD, Jones CA, Tompkins SM, Tripp RA. One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst. 2011;136(15):3083-3090.

Jonathan Faro, MD, PhD, is an Assistant Professor in the Department of Obstetrics, Gynecology and Reproductive Sciences at the University of Texas Health Sciences Center, Houston. His research interests include rapid diagnostics and their clinical applications. With financial support from NanoLogix, Inc., he has helped to develop an assay for group Bstreptococcus.

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