Identification of Microbial Pathogens Using Nucleic Acid Sequencing

By Peter C. Iwen, PhD, Associate Director, NPHL For more than 100 years, Robert Koch’s postulate that required in part the cultivation of a pathogen to show a disease/pathogen relationship, was seldom questioned and was considered the basic standard used in clinical diagnostics. Organism identification to taxon (species, genus) was subsequently accomplished by studying phenotypic characteristics such as Gram stain, morphology, culture requirements, and biochemical reactions along with a combination of intuition and stepwise analysis of the results. In today’s laboratory, the ability to detect and identify pathogens has undergone major changes. The development of molecular methods that rely on the detection of genomic elements (DNA or RNA) with or without culture has led the way in this charge. Some of the main reasons for this change from phenotypic to molecular testing include such issues as the slow growth of pathogens, the detection of organisms that exhibit biochemical characteristics that do not fit patterns of known species, and the inability to detect non-cultivatable organisms. Although culture-based methods are still considered the gold standard for identification diagnosis, molecular methods have emerged as the confirmatory method for identification in many diagnostic applications. The basic principle of any molecular test is the detection of a specific nucleotide sequence (signature sequence) within the organisms’ genome which is then hybridized to a labeled complementary sequence followed by a detection mechanism. The first application of these methods in the clinical laboratory was in the development of labeled probes for culture confirmation testing. The original probes were designed to detect “problem” pathogens such as those that were historically difficult to identify using phenotypic methods. These original probes included tests for the culture confirmation of dimorphic fungal pathogens (Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum) and to identify the more common Mycobacterium species (M. tuberculosis complex and M. avium complex). Subsequently, direct detection probes were designed for high volume testing of STD pathogens e.g., Chlamydia trachomatis and Neisseria gonorrhoeae and for the testing of pathogens that were difficult to grow and identify in the laboratory e.g., Legionella pneumophilia and Human papillomavirus. Although extensively used today, nucleic acid probing unfortunately has been shown to have limited selectivity and to lack sensitivity when testing from direct specimens. To overcome these problems, a process whereby the genomic target could be amplified using non-selective means was developed. The most widely used method for nucleic acid amplification is the polymerase chain reaction assay i.e., PCR. This assay includes a specific primer pair to amplify a unique genomic target nucleotide sequence for analysis. Following PCR, a variety of post-amplification methods are used to evaluate the product such as direct sequence analysis, use of genus or species specific probes, and utilization of restriction enzymatic analysis of the product, e.g., restriction fragment length polymorphism analysis (RFLP). Even though all these post-amplification methods have been shown to be useful for the evaluation of microbes, sequence analysis is considered a particularly useful method for the identification of microbial species due to its wide range application to a variety of species. The basic steps involved in this technique are shown in Table 1. One drawback to this methodology is that access to sequencing facilities is not readily available for many laboratories, limiting the ability of most laboratories to conduct routine sequence analysis testing. To overcome this issue, commercial kits and some reference laboratories now offer lowcost sequencing for those instances where identification is required for diagnostic purposes.