Clinical microbiology laboratories were central to the tough but successful fight against infectious diseases in the 19th and first half of the 20th centuries, and resonate in the names of now-iconic figures from Jenner, Pasteur and Lister to Koch, Gram and Fleming.
In the 1980s and 1990s, breakthroughs in biotechnology, especially DNA sequencing and genomics, arrived as game-changers. Over 1,000 bacterial genomes and 3,000 viral genomes, including representatives of all significant human pathogens, have already been sequenced. Coupled with novel proteomic analyses, this has led to unprecedented advances in pathogen diagnosis and genotyping and in the detection of virulence and antibiotic resistance.
Together with high-speed computing and automation, increasingly sophisticated genomic tools now play a central role in the clinical microbiology laboratory and seem set to continue shaping its role in the 21st century.
Tough new strains of antibiotic resistant bacteria
The need for a sophisticated arsenal of technological tools in the microbiology lab is dictated by two recent developments. The first is the emergence and growth of tough strains of microbes resistant to antibiotics, even as the pipeline of new antibiotics is drying up. Microorganisms such as K. pneumoniae and E. coli have become resistant to third-generation cephalosporins as well as carbapenems. In April 2011, the World Health Organization (WHO) warned that antibiotic-resistant superbugs threaten to take us to the pre-penicillin era, where even the smallest infection could potentially be fatal. Resistant bacterial strains are also pervasive. In Holland, 94% of a representative sample of chicken meat at retailers was found to be contaminated with resistant E. coli isolates. In June 2011, 70 global experts, including many clinical microbiologists, met in France to issue the so-called Pensières Antibiotic Resistance Call to Action, which seeks a coordinated worldwide response to the challenge.
Globalization: the need for speed
The second factor is globalization, which has sharply accelerated the numbers of people travelling across frontiers, and sometimes carrying diseases. The number of international tourists grew from 69 million in 1960 to 278 million in 1980 and 687 million in 2000. By 2012, the number had crossed 1 billion. This trend is of specific concern for viral outbreaks of respiratory diseases such as SARS and avian flu. It also has relevance for certain bacterial conditions such as salmonella, which has witnessed the growth of antibiotic-resistant strains and is estimated to result “in more than hundred thousand deaths” a year.
New diagnostic tools in the lab, therefore, have to be fast. Traditional techniques, based on culturing the pathogenic agent, often take several days to provide results.
Diagnostic systems also need to scale up quickly, should mass screening be required. Unfortunately, this carries a price. In the case of influenza, for instance, point-of-care enzyme immunoassays (EIAs) can provide results in 15 minutes. However, they still have considerable potential for false negatives.
The promise of molecular technologies
Several emerging molecular array techniques hold the potential for clinical microbiologists to meet the twin challenges of speed and scale, and to do so with accuracy. Above all, these consist of direct nucleic acid detection techniques, high-throughput nucleic acid extraction techniques and next-generation sequencing. These, in turn, are coupled with automated platforms, which provide labs with a capability to perform on-demand testing, in increasingly shorter turnaround times. The focus of researchers now is to move beyond detecting single-analytes to multiplex targets and detect more pathogens from a single specimen.
These new techniques have been enthusiastically adopted, in particular by virologists, and have almost entirely replaced traditional viral tube culturing. Indeed, only modest improvements in culture-based systems have been made over many years, and they are simply not enough for sustainability in this century and beyond. On the other hand, molecular methods have led to discoveries never before imagined.
Direct nucleic acid detection
Direct nucleic acid detection is based on supplementing polymerase chain reaction (PCR) amplification technology with an enzyme reverse transcriptase step (RT), in order to trace viral RNA at low levels. New molecular assays such as nucleic acid amplification techniques (NAAT), which use a series of repeated reactions to make numerous copies of the DNA or RNA, allow for greater degrees of analytical sensitivity and specificity than PCR. The latest generation of tests can detect infectious agents during their incubation period and hold forth the capability to identify any virus.
High-throughput magnetic bead extraction
High-throughput nucleic acid extraction adds more ammunition to the lab arsenal, with faster, cleaner and more consistent results. State-of-the-art extraction technologies are increasingly based on magnetic beads rather than vacuum/silica filtration, which entails longer processing times and volume limitations, and also complicates integration with automated systems. Some magnetic bead extraction systems (such as PerkinElmer’s chemagic MSM1) dramatically enhance flexibility in sample handling, expanding maximum input volume levels from 500 or 1,000 µl to 10,000 µl, while reducing minimum levels from 100 or 200 µl to 10 µl. They also increase precision by eliminating the need for pipetting, and have features such as cover plates to reduce contamination. Finally, such systems are designed to directly interface with LIMS rather than PCs, CDs or USBs. The chemagic MSM1 allows for almost 2,500 tests per 8-hour day from 200-500 in typical vacuum extraction systems. However, most magnetic bead-based systems retain lower throughputs, and base their USPs on heightened pipetting efficiency (below 2% compared to the 4-5% in vacuum technologies) as well as lower sample input requirements. On another front, certain vendors have developed systems such as the BioRobot Universal, which can be based on either vacuum or magnetic bead technology.
Next generation sequencing
Next-generation sequencing (NGS) is another tool for highly multiplexed tests on infectious disease. It is a follow-on to capillary-electrophoresis (CE)-based Sanger sequencing, which has been adopted by labs around the world but has been hampered by inherent limitations in throughput, scalability, speed and resolution. The underlying principle of NGS is similar to CE, by which DNA fragments are identified by signals as they are resynthesized from a template strand. However, while CE is limited to one or a few DNA fragments, NGS uses massively parallel processes to cover millions. This allows for identification of DNA base pairs covering whole genomes.
The latest NGS systems can generate up to 300 Gb per flow cell, discover SNPs and chromosomal rearrangements, conduct transcriptome analysis, generate expression profiles, detect splice variants and quantify protein-DNA interactions.
One key application of NGS is the rapid detection of viral resistance.
Another is diagnostic. For example, the market for DNA-sequencing-based tests for Down Syndrome has recently been estimated at upwards of $6 billion.
Molecular diagnostics also has immense potential in bacteriology, by allowing for identification, antibiotic resistance tests, and the monitoring of pathogen growth. Adoption is being led in instances where their twin advantage, of speed and scale, are especially pronounced. One application of molecular diagnostic tests are in hospital-acquired infections (HAI), especially Clostridium difficile and septicemia.
A widely-cited animal study on antibiotic treatment for E. coli septic shock found an inflexion zone of 12-15 hours in terms of survival. Treatment initiated before 12 hours showed a 20% mortality rate, but this rose sharply to 85% after 15 hours. Rapid diagnostics therefore seems to be a critical factor for bloodstream infections.
Hospital acquired C. difficile has also been increasing dramatically in recent years, with molecular assays commercially available for identification and detection as well as corresponding antibiotic resistance markers.
The use of more efficient molecular diagnostics has also driven development of tests against diseases with prospectively large scales of infection.
Indeed, the first NAATs to be approved in the USA were for detecting Chlamydia trachomatis, as far back as 1993. Since then, more sophisticated NAATs for chlamydia as well as other STDs such as gonorrhoea have minimized inhibition from sample components and improved workflow.
Molecular tests look especially promising for tuberculosis mycobacterium testing, where whole-genome sequencing is believed to have equivalent cost-effectiveness as MTBDR assays, but also provides species identification and resistance determinants. In 2008, the Association of Public Health Laboratories and the US Centers for Disease Control recommended NAATs as standard practice to aid in the initial diagnosis of patients with suspected TB, and in August 2013 the FDA approved its first NAAT for rifampin-resistant strains.
NAATs: New weapon in surveillance arsenal
The final driver is surveillance. NAATs are already recommended by the WHO for drug-resistant TB. A number of health agencies, including Britain’s, are exploring the adoption of whole-genome sequencing to eventually implement it “as the preferred typing method for outbreak investigation and pathogen surveillance.” One powerful argument in favour of molecular tests here is that “in principle, detection of an outbreak could occur as early as the first secondary case.”
Cost and regulations remain barrier
However, there are some obstacles to the growth of molecular diagnostics in bacteriology. One of the biggest is cost. Molecular testing is not yet considered affordable by smaller laboratories.
A more serious problem pertains to the regulatory context of a microbiology laboratory.
The European Society for Clinical Microbiology and Infectious Diseases laments that the two specialties “have different training requirements” in different EU countries, and, worse, are not even “universally recognized as professions.” Evidently, this poses some problem for the adoption of expensive, cutting-edge lab technologies targeted at infectious diseases.
Across the Atlantic, the American College of Microbiology notes that despite “the pivotal role of clinical microbiology laboratories in our healthcare system,” they “are victims of difficult times.” In an assessment of the lab’s role in the 21st century, the College highlights difficulties “in translating promising research achievements into tangible improvements” in the fight against infectious diseases. The main reason for this, it notes, is a lack in the number of laboratories with expertise “to perform increasingly complicated laboratory tests,” especially “in the area of molecular diagnostics.”