The Molecular Edge
Molecular methods have been slow to be adapted into microbiology. Up to this time, the announcements of molecular methods and polymerase chain reaction (PCR) have not reached the anticipated impact in microbiology or in other rapid methods of detection and recognition.
Molecular methods have been applied mostly to virology, although the applications in some areas for screening have had impact, particularly with MRSA. Microbiologists are well-equipped for the coming of the molecular age. There is no doubt that molecular techniques will revolutionize the practice of medical microbiology, but can there still be a place for Gram stains and culture plates once molecular has taken over?
The answer to that question is multifaceted. Medical microbiology has embraced the molecular age and has essentially adopted it for the use of everyday diagnostics. Furthermore, there are many advantages to the use of molecular technology in the lab over the continuation of culture techniques. Many jobs in the microbiology laboratory are now being performed on high-throughput instruments, but almost every operation still requires the technician to culture microorganisms, especially in the food-service industry. Whether testing is aimed at detecting pathogens, counting potential spoilage organisms, studying the metabolism of bacteria and molds or identifying a contaminant, the first requirement is almost always to culture the organism so there are enough cells to detect or study.1
The problem with having to culture microorganisms as part of the process is that the overall result time is significantly increased. In many cases, waiting for the culture to grow adds at least 24 hours to a test. The reliance on culture limits how soon the results can be related to the patient as well as the ability to grow certain types of organisms.
There is no doubt that PCR has revolutionized medicine, particularly molecular medicine, but is there a place for it to be used in microbiology? The answer is a resounding yes. This strongest challenge yet to traditional diagnostics has its roots in the 1980s with the development of molecular biology techniques focusing on the genes of microorganisms, rather than their physical and metabolic characteristics. The key discovery was the PCR, first developed in 1983 by Kary Mullis, MD. Dr. Mullis took the rather simple steps for copying DNA and made it possible for us to make tons of exact replicas.
Just as the equipment has changed over the years, so have the techniques. The use of PCR has made it so easy to copy DNA that high school students are able to perform these experiments in their classrooms-all within the space of a day, and even a class period. The methodology is straightforward and is practical enough to use in the diagnostic medical laboratory.
Additional exciting advancements include terminal restriction fragment length polymorphism (T-RFLP) analysis, next-generation sequencing, PCR-mass spectroscopy and IVIS imaging. These are more evolved and novel and may be better candidates to usurp the position currently held by microbiological culture.
Furthermore, direct sequencing from patient samples will address rapid detection of endocarditis, bone and joint infection in a timely manner that improves the outcome for patients. Targets like 16-SRNA and the detection of diverse co-existent microbial populations are much sought after endpoints.
Does Culture Have a Place?
The development of real-time PCR has moved microbial diagnostics into the next level of accuracy. In doing so, real-time PCR has driven significant changes in the way we detect microbes. We can now use real-time PCR to detect and quantify microbial nucleic acids. Where we used to use only our eyes to visualize the phenotypic differences in cultures, we can now know more about the offending organisms and in astoundingly less time. Real-time PCR has engendered wider acceptance of the PCR technique due to its improved rapidity, sensitivity, reproducibility and the considerably reduced risk of carry-over contamination.2
The majority of real-time PCR applications used today in microbiology are for qualitative detection of a virus, bacterium, fungus or parasite. In terms of disease relevance, the importance of quantitative PCR (qPCR) to microbiology has been proven; however, it is less clear just how thorough the clinical microbiology laboratory must be to produce results that will mean something of value to clinicians. Furthermore, will doctors use that detailed information to change their treatment strategy? It is hoped that we will be able to determine not only the particular microorganism that a patient may have, but also what stage it is in and how the patient's immune system is responding to the infection.
To make such vision reality, we must first discuss and reach consensus on the best microbiology-specific qPCR approaches to permit the production of reliable and reproducible microbial load data. We may find that the actual absolute number of organisms isn't as critical as clinically relevant data. The increased identification of newly emergent or previously unknown endemic pathogens demands that we must strive harder than ever to expand our understanding of infectious diseases, and for that we need reliable results from reliable tools that are available to most of us.
The Final Frontier?
As we move further from the use of time-consuming culture methods in the classic microbiologic lab, we are seeing the value and ease of molecular methods. We should be focusing our energies into developing even more affordable and practical molecular tests.
Mixtures of amplicons and T-RFLP profiling have had significant impact in environmental samples, oral flora and environmental sampling best associated with multiple species. Next-generation sequencing and/or high-throughput sequencing has the power for amplification of genetic material by PCR, then using ligation of amplified material to a solid surface. Sequencing is done in a massive parallel fashion; sequence information is captured by unique software. This will dramatically change the landscape for clinical implementation of molecular microbiology.
Dr. Mandes Wildemore is a Community College of Philadelphia visiting lecturer.
1. Lawle y R. (2010) A revolution in the microbiology laboratory. http://www.foodsafetywatch.com/public/1050.cfm. Last accessed December 19, 2010.
2. Mackay I. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, chap 7.
3. Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002 Nov;81(11):761-6.