The process of culture-based testing for microbes has been in common use for more than a century. In recent decades, molecular diagnostic tests have emerged as a more specific, sensitive, and faster alternative. In many cases, however, these tests require multimillion dollar molecular labs and highly skilled technicians to run them. Thus, while molecular diagnostic techniques can theoretically generate more sensitive and specific reactions in hours instead of the days needed for a culture-based test, they have remained out of reach for many organizations.
The three key steps to performing a real-time polymerase chain reaction (PCR)—sample preparation and extraction of nucleic acids, amplification of extracted nucleic acids, and detection of a target gene sequence—were first developed in the 1980s. These steps are time-consuming and are not amenable to on-demand testing. Institutions fortunate enough to have a molecular lab typically process samples in batches.
First-generation PCR assays for molecular diagnostics required manual sample extraction using reagents prepared by the laboratory itself and often involved enzymatic digestion, extraction with organic solvents, and alcohol precipitation. After drying down the nucleic acid pellet, it was then resuspended and added to a custom, laboratory-prepared buffered cocktail of enzymes, nucleotides, and oligonucleotide primers for carrying out the PCR process. After amplification, the real work of detecting the amplification products began. For best sensitivity, labeled probes were used to detect PCR amplification products on Southern blots or in microtiter plates. The two-to-three day process of carrying out the complete diagnostic procedure was labor intensive and error prone, primarily from laboratory contamination, which gave rise to false positive results.
The next milestone in the development of diagnostic applications of PCR was the development of real-time PCR assays. These assays use labeled probes that are part of the initial PCR reaction mixture; detection of fluorescence accumulation during thermal cycling is used in place of Southern blots for detection of PCR products within the closed environment reaction tube. Real-time PCR is now the most commonly used method for user-developed assays, although only a few commercial kits incorporate this technology.
Still to be refined, however, has been the sample-preparation component. No universal method for sample preparation exists. Although several kit-based methods are adaptable to a variety of specimens, these methods are still not well adapted to on-demand testing.
This paradigm has now shifted, ushering in a new era in how tests are processed, data is analyzed, and patient care delivered. Today, advances in molecular diagnostics and the ability to automate molecular reactions have the power to move the lab to the front lines of medicine more efficiently and cost-effectively. The lab is democratized as new molecular diagnostic technologies now bring the same benefits to a broad spectrum of facilities: large reference labs, regional hospitals, field research, and first response teams.
The key is automation, which now makes it possible for virtually anyone to collect a sample and carry out a molecular reaction. This is a major step in applying the technology to a growing list of public health priorities.
Practical Uses for Automation
Take, for example, the threat of methicilin-resistant Staphylococcus aureus (MRSA). A study published in the December 1, 2006, issue of The Journal of Infectious Diseases found that 90 million Americans were colonized with staph bacteria, while two million Americans were colonized with the antibiotic-resistant MRSA strain. Another study published in the February 1, 2006, issue of Clinical Infectious Diseases found that from 1992–2003 staph infections contracted in hospitals rose from 35.9 to 64.4%.
In a healthcare setting, MRSA is spread among doctors, patients, nurses, and visitors who come in contact with contaminated surfaces like bed rails or computer keyboards. If MRSA enters the body through the skin, it can cause irritating skin infections but if it enters the bloodstream or lungs, it can cause serious blood infections, pneumonia, and even death.
Studies show that diligent surveillance programs, where patients are screened upon admission, have a tremendous impact in reducing MRSA infection rates. Identifying MRSA carriers, isolating them, and administering antibiotics is an effective method to stop the spread of MRSA. In fact, the Netherlands, Sweden, and Denmark have virtually eliminated MRSA from their hospitals with stringent surveillance programs.
Culture-based testing takes days to produce a result—it is too slow to be highly effective in isolating carriers from non-carriers. Enter automated molecular diagnostics that enable hospitals to collect a specimen with a nasal swab and process a test in about an hour. In addition to reducing the spread of MRSA and improving patient outcomes, surveillance presents significant cost savings—un-reimbursed costs of treating a single MRSA infection average $25,000–40,000. A growing number of American hospitals are thus adopting MRSA surveillance programs. Better tests for automated molecular diagnostic instruments make the technology even more accessible and affordable.
MRSA is a good example of how automated molecular diagnostics meet an important demand for improving hospital surveillance modalities. The technology also offers tremendous benefits by providing critical information at the point of care such as emergency or delivery rooms.
According to the CDC, group B Streptococcus (GBS) bacterium is the most common cause of life-threatening infections in newborns and is the leading infectious cause of neonatal morbidity and mortality. Untreated, GBS can cause the development of sepsis, pneumonia, and meningitis—leading to sight or hearing loss, mental retardation, or death. Treatment of infected mothers and infants cost the healthcare system approximately $300 million each year.
Most women are screened for GBS during their antepartum period, 35–37 weeks gestation. However, several studies have shown that screening results can change quickly—what if a woman tests negative for GBS yet becomes a carrier after 37 weeks? What about the woman who does not receive regular prenatal care and arrives at the hospital in labor with an unknown GBS status?
Today, an automated molecular diagnostic test can be performed during the intrapartum period. With culture-based testing, a rapid result was impossible, but today, an obstetrician can get the results of a molecular GBS test in about an hour, enabling successful delivery without concern or the use of unnecessary antibiotics.
The Laboratory's New Role
These are just two of the many examples that demonstrate the benefits of bringing molecular diagnostics to the front lines of healthcare through advances in molecular automation. Think of the benefit this technology offers for monitoring the spread of the H5N1 virus to combat a bird flu epidemic. Consider the role molecular diagnostics could bring to developing nations where a rapid diagnostic test for tuberculosis could play a major role in treating HIV-infected populations.
The next step in the evolution of molecular diagnostics is to expand the menu of tests that support new automated instruments. As new tests become available, the industry will see molecular diagnostics making a greater and greater impact in clinical settings. Ultimately, this will take the lab out from behind closed doors, making it available to clinicians 24/7.