The X-games of PCR

This is not your Mom’s PCR. These new kids on the block are making PCR extremely fast. PCR (Polymerase Chain Reaction) technology won the Nobel Prize for allowing molecular research to advance much more rapidly (for an interesting read on the quirky Laureate who gave up science to go surfing, read more here: Wikipedia ). It has become the most commonly used work horse of most molecular diagnostic assays, usually in the form of real-time PCR. It is used for a variety of purposes from detecting bacteria and viruses, identity testing for forensics and bone marrow engraftment, cancer mutation analysis, and even sequencing by synthesis used by Illumina for massively parallel sequencing.

This technique is still limited by requiring highly trained technologists to perform DNA extraction, time-consuming processing, and the time of real-time PCR itself. Overall, this process takes about a 5-8 hours. While this is much faster than in the past, it would be unacceptable for use in the point-of-care (POC).

But why would DNA testing need to be POC? The term sounds like an oxymoron in a field where many results have a 2-month turnaround time. There are certain circumstances where molecular testing would impact patient care. For instance, a doctor testing a patient in their office for a sexually transmitted infection would want to know if they have gonorrhea/ chlamydia so they could prescribe proper antibiotics. Similarly, POC molecular testing could be applied in a bioterrorism incident to test samples for an infectious agent. Or POC testing would benefit low-resource areas internationally where HIV testing could be used to manage anti-retroviral therapy in patients many miles from a laboratory.

For PCR as a test to be useful at the POC setting, it would have to provide a result within 10-15 minutes and be performed as a waived test. Two recent examples the demonstrate how this is possible have been highlighted at recent conferences of the American Association of Clinical Chemistry, which I just got back from: Extreme PCR1 and Laser-PCR.2

Extreme PCR refers to a technique of rapidly cycling the temperature of PCR reactions. The reaction occurs in a thin slide that evenly distributes the reagents, temperature and is clear to permit easy reading of fluorescence measurements (Figure 1). DNA Polymerase enzyme and primers to amplify the target DNA are added at much higher concentrations than normal (20x).

Figure 1. Thin reaction chamber for ultra-fast PCR.

This flies in the face of traditional PCR chemistry dogma as specificity would plummet and normal DNA could be amplified instead of target DNA. This would create a false positive. However, let’s think about what is actually happening with non-specific reactions. Primers are designed to match one region of DNA, which is very unique within the whole genome. However, the genome is so large that some segment may look very similar and be different in just 1 or 2 of the 20 base pairs that a primer matches. A primer could bind to this alternate region but less efficiently. So, the binding would be weaker and take more time to occur.

Therefore, by speeding up the cycling time to just a few seconds, only the most specific interactions can take place and non-specific binding is offset (Figure 2)!

Figure 2. Fluorescence from a dye that fluoresces when bound to double stranded DNA, which is increasing here within seconds (high point represents when the reaction temperature cools and dsDNA anneals, then low points represent heating to high temperatures).

Laser PCR does not report the use of increased reagents like Extreme PCR (it may be proprietary), but they boast a very innovative method to quickly heat and cool PCR reactions. GNA Biosciences use gold nanoparticles with many DNA adapters attached (Watch the video below for a great visual explanation!).

These adapters are short sequences of DNA that bring the target DNA and primers together to amplify the target DNA sequence. Then as the name implies, a laser zaps the gold beads and heats them up in a very localized area that releases the DNA strands. The released DNA binds another gold particle, replicates, rinses, and repeats. The laser energy thus heats the gold in a small area that allows for quick heating and cooling within a matter of seconds.

These new PCR methods are very interesting and can have a big impact on changing how molecular pathology advances are brought to the patient. On a scientific note, I hope you found them as fascinating as I did!

References

  1. Myrick JT, Pryor RJ, Palais RA, Ison SJ, Sanford L, Dwight ZL, et al. Integrated extreme real-time PCR and high-speed melting analysis in 52 to 87 seconds. Clin Chem 2019;65:263–71.
  2. CLN Stat. A Celebration of Innovation. AACC’s first disruptive technology award to recognize three breakthrough diagnostics. https://www.aacc.org/publications/cln/cln-stat/2018/july/10/a-celebration-of-innovation
  3. G. Mike Makrigiorgos. Extreme PCR Meets High-Speed Melting: A Step Closer to Molecular Diagnostics “While You Wait” Clin Chem 2019.

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

When Rapid Blood Culture Identification Results Don’t Correlate, Part 1: Clinical Correlation Needed

More and more laboratories perform rapid (i.e., multiplex PCR) blood culture identification. For the most part, it has been a wonderful addition to the laboratory workflow, not to mention the added benefits of provider satisfaction and improved patient care. Because the PCR only provides the organism identification (sometimes only to the family-level, i.e.; Enterobacteriaceae), laboratories must continue to culture the positive blood for definitive identification and/or antimicrobial susceptibility results. So what do you do when the results don’t correlate?

The Issue

From time to time, the PCR result is not going to correlate with the direct Gram stain or with the culture results. Although this is an issue one would fully anticipate, what do you do when this happens? Do you take some sort of action to arbitrate? Do you report the results as is?

First of all, the PCR assays do not detect all organisms. They only detect the most common bloodstream pathogens. Therefore, one should fully expect to observe cases in which the Gram stain would be positive, but the PCR results would be negative (scenario 1).  This is not a surprise.

Additionally, one should also assume that the PCR will occasionally detect organisms that were present at the lower limit of detection of the Gram stain. An example of this would be that the Gram stain is positive for one morphology (i.e.; Gram-positive cocci), but the PCR is positive for two organisms (i.e.; Staphylococcus and a Proteus species). Most of these cases tend to correlate with culture. In other words, although the second organism was not originally observed in the Gram stain, it was detected via PCR and then it also subsequently grew in culture (scenario 2).

Another type of discordant result laboratories sometimes experience is when the organism detected via PCR does not grow in culture for whatever reason. Similar to scenario 2 stated above, except that the culture is also negative for the second organism (scenario 3). Perhaps the patient was treated with antibiotics and the organism is no longer viable for culture? Perhaps a sampling or processing error was to blame?

The Solution

Depending on the scenario and how much work you want to do, you can either repeat testing or try an alternative method. Take scenario 2 for example. If the PCR detects two organisms and the Gram stain is only positive for one, then review of the original Gram stain is warranted. It is possible that the Gram-negative was somehow missed. Our eyes tend to go to the darker, more obvious structures. Perhaps the Gram-negative organism was faintly stained and it was overlooked? It is also possible that the Gram-positive is present in much lower numbers and only Gram-negative organism was originally observed. If the Gram stain result remains the same after review (only one organism observed), then there is nothing much left to do except to wait for the culture. That being said, an alternative method, such as acridine orange can be utilized in this type of scenario (two different cell morphologies). Acridine orange is a fluorescent stain that improves organism detection, as it is more sensitive than the Gram stain (1, 2).

If only the Proteus is growing (and the Staphylococcus isn’t from scenario 2) and we normally subculture positive blood to blood, chocolate, and MacConkey agars, then perhaps including an additional media that inhibits Gram-negative growth would be beneficial.

Scenario 3 can be a little more difficult to solve because you can’t make a non-viable organism grow. It just is what it is. [Spoiler alert: in next month’s blog I plan to write about when you should change your thinking from true-positive to false-positive.]

Regardless of why the result is discrepant, our laboratory appends a comment to the discordant result which says, “Clinical correlation needed.” This lets the clinician know that the results are abnormal and that they must use other relevant information to make a definitive diagnosis. In addition to the comment, we also make sure the discrepancy is notified to laboratory technical leadership (i.e.; Doctoral Director, Technical Lead/Specialist). This allows us to keep track of discrepancies as they may become important to know about in the future (see next month’s blog).

The Conclusion

In terms of organism detection, nucleic assays (i.e., NAATs) can provide superior sensitivity over antigen and culture-based methods of organism detection (i.e., sensitivity = PCR > culture > Gram). From the laboratory perspective, other potential benefits of utilizing nucleic acid detection methodologies include decreased TAT, simplified workflows, and reduced hands-on time. In terms of patient care, many have noted improved outcomes due to increased sensitivity and decreased time to result.

Although advances in technology can significantly improve analytical performance, they can also add complexity to the post-analytical process. Making sense of the results can sometimes lead to confusion. It is important to know the product’s limitations and what your risk(s) is. This should already be known and included in your Individualized Quality Control Plan (IQCP). Lastly, guiding the clinician to proper result interpretation is also important to maintain valuable patient care.
References

  1. Mirrett, S., Lauer, B.A., Miller, G.A., Reller, L.B. 1981. Comparison of Acridine Orange, Methylene Blue, and Gram Stains for Blood Cultures. J. Clin. Microbiol. 15(4): 562-566.
  2. Lauer, B.A., Reller, L.B., and Mirrett, S. 1981. Comparison of Acridine Orange and Gram Stains for Detection of Microorganisms in Cerebrospinal Fluid and Other Clinical Specimens. J. Clin. Microbiol. 14(2): 201-205.

 

Martinez Headshot-small 2017

-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.