Within the past decade, clinical microbiology laboratories have experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Timely and accurate identification of microorganisms is the underlying function of any clinical microbiology laboratory and it is accomplished through a consistently evolving repertoire of laboratory techniques. The microbiology instruments and reagents have been an indispensable part of routine in most clinical laboratories and diagnostic centers.
The microbiology market is growing due to increase in prevalence of pathogenic diseases, growth in discovery of mutating and adapting bacteria, and the growing need for speedy microbiological testing methods. Thus, clinical laboratories are under immense and relentless pressure to improve turnaround times, reduce human error rates, improve efficiency, and make cost savings.
The clinical microbiology laboratory plays a central part in optimizing the management of infectious diseases and surveying local and global epidemiology. This pivotal role is made possible by the adoption of rapid diagnostic techniques, rational sampling, point-of-care (POC) tests, extended automation, and new technologies, including mass spectrometry for colony identification, real-time genomics for isolate characterization, and versatile and permissive culture systems. When balanced with cost, these developments can improve the workflow and output of clinical microbiology laboratories and by identifying and characterizing microbial pathogens, provide significant input to scientific discovery.
Rapid diagnostic identification and characterization of infectious pathogens are essential to guide therapy, to predict outcomes, and to detect transmission events or treatment failures. Current clinical microbial diagnostic methods are mainly based on conventional culturing of clinical samples on different agar plates, followed by susceptibility testing and further characterization on a case-by-case basis. Several methods for rapid diagnostic testing directly with clinical samples have been developed and evaluated, including polymerase chain reaction (PCR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). These technologies, however, do not give information beyond species identification.
In recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a potential tool for microbial identification and diagnosis. MALDI-TOF MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting rapid and robust method for accurate microbial identification. Multiple instrument platforms, marketed by â€¨well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices.
Real-time PCR and fluorescence in situ hybridization (FISH) methods have established new norms for speed and sensitivity.
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. The use of real-time PCR for molecular diagnostic applications requires a high degree of assurance that the analytic result reflects accurately the true concentration of the target nucleic acid and is not affected by inhibitors of the reaction. A variety of strategies have been developed to ensure confidence in the resulting assay information. Capitalizing on the strengths of rtPCR, we can further expand the capabilities of the methodology, so as to detect multiple target nucleic acid sequences in a single reaction.
While multiplexing of rtPCR assays is achieved on a limited basis - typically two to four target sequences - there are examples of highly multiplexed assays such as DNA arrays or gene chips that can interrogate >10,000 oligonucleotide sequences in a single sample. Through integration of multiple assays into a single reaction, information about assay quality (e.g., internal positive or inhibition controls) can be simultaneously generated, and additional target pathogen sequences queried. Multiplexing, therefore, reduces analytical costs, improves turnaround time, expands testing capability and capacity, and adds data richness to analyses.
The age of reliance upon in vitro cell culture for routine laboratory diagnosis of respiratory virus infections has well and truly passed. The increased acceptance of molecular tools is not at the complete expense of other biological or serological methods; they will always have a place in microbiology; however the era of the â€¨high-throughput laboratory has driven the use of faster, more sensitive, and more specific methods to diagnose viral pathogens. Unfortunately, these methods have some inherent limitations and the use of molecular techniques pose a number of serious problems to overcome, especially apparent in the area of respiratory virus detection and characterization. The scope and diversity of respiratory viruses mean that scientists in this field have to design and evaluate their own assays in-house because commercial options are extremely limited.
One major shortcoming of most currently available rapid diagnostic testing is their limited information on antimicrobial susceptibility. While some of these technologies are able to identify resistance markers, which are helpful in particular instances such as MRSA and GRE, full antimicrobial susceptibility data is not yet readily available.
Real-time PCR cannot be used in isolation; for maximum success it must be accompanied by nucleotide sequencing, phylogenetic analyses, and constant vigilance over the relevant literature. There is also a driving need to delve into the increased co-detection of multiple viral sequences and the ill-defined impact of quasi species variation on oligonucleotide design.
Another limitation of currently available rapid diagnostic testing methods to evaluate patients with presumed bacteremia is that they can only be performed after a culture system has detected microbial growth. The future of rapid diagnosis would be to look at a syndromic approach and tailor the tests accordingly. Improving the diagnostic accuracy of tests should be a goal of the manufacturers and education of personnel on the use and interpretation of these tests must be in the domain of microbiologists.