Microbiology Laboratory Transformation From Conventional Methods To Molecular Technologies
Microbiology laboratories play an important role in epidemiology and infection control programs. Within microbiology laboratories, molecular microbiology techniques have revolutionized the identification and surveillance of infectious diseases. The combination of excellent sensitivity, specificity, low contamination levels, and speed has made molecular techniques appealing methods for the diagnosis of many infectious diseases. A high-quality laboratory should include both conventional microbiology methods and molecular microbiology techniques for exceptional performance. Furthermore, it should include appropriate laboratory administration, a well-designed facility, laboratory procedure, standardization, a waste management system, a code of practice, equipment installation, and laboratory personnel training.
Molecular techniques in high-quality clinical microbiological laboratories are routinely used to make clinical decisions on the basis of how and when to treat patients. Molecular techniques are also useful for monitoring the effectiveness of therapeutic regimes and identifying potential resistant strains that may impact a long-term treatment program. Conventionally, the function of a clinical microbiology laboratory is to identify various pathogens using direct examination of microbes and culture-based methods. At present, nucleic acid amplification and DNA probes are useful for the characterization of microbes because culture-based methods are insufficient, expensive, and tedious. Typically, non-amplified DNA probe-based methods are suitable for in situ hybridization of organisms when the location and distribution are ascertained, including slow growing microbes.
Many DNA probe methods are commercially available for the identification of various microbial species.
New molecular technologies
Real-time droplet PCR. Because PCR requires thermal cycling, it is typically performed in a bench top unit. This process is slow, expensive, and error-prone due to contamination and requires a large amount of sample to perform the reactions. Utilizing the concept of both spatial and temporal micro fluidic PCR, droplet-based micro fluidic PCR formats are emerging as alternatives to single-phase PCR. Droplet PCR formats have many advantages, such as low reagent usage, faster thermal cycling, and limited interactions of DNA and polymerase with the channel walls. Carryover contamination can be reduced using these formats, resulting in high throughput and sensitivity.
Mass spectrometry. Based on MS, MALDI coupled with time of flight (TOF) allows the analysis of various biomolecules in microbes. This method is very effective and requires less intense sample preparation because the matrix is less susceptible to interference from salts and detergents. MALDI-TOF is an extremely useful technique for the identification of many microbial species because the major mass profile is generated from ribosomal proteins that align perfectly with current taxonomic classifications. Cellular modifications under the influence of antibiotics can also be detected, as was recently demonstrated by the interaction between Candida albicans cells and Fluconazole and Candida and Aspergillus species and caspofungin.
It is important to remember that despite their sensitivity, accuracy, and speed, nucleic acid amplification methods cannot totally replace conventional culture. Nucleic acid amplification methods detect the presence of DNA or RNA in a particular specimen, but they cannot demonstrate the viability of an organism or determine whether the organism is part of the infectious process. Conventional methods such as culture-based techniques validate the viability of an organism, and quantification of the antibody titer strongly suggests its involvement as part of the infectious process.
