The field of molecular diagnostics is dynamic and rapidly evolving. The methods described below have been adopted by clinical microbiology labs not only due to their increased sensitivity and specificity and reduced turnaround time, as compared with more traditional diagnostic methods, but also because early and accurate diagnosis has a significant and positive impact on patient care. It is not the intent here to discuss all assays that are currently available or under development. Rather, it is important for clinicians to be aware of the pace of development for these molecular techniques and tools and to consult with the clinical microbiology lab when considering assays that are most appropriate for any given patient.
Genomic Tests
Molecular diagnostic tests can be broadly categorized into those that evaluate nucleic acids (DNA or RNA) and those that assay for proteins or enzymatic activity. There are three types of nucleic acid–based tests used in the diagnosis of bacterial diseases: NAATs (nucleic acid amplification tests), nucleic acid probes, and nucleic acid sequence analysis; many of these tests have become a routine part of clinical microbiology diagnostics.
Nucleic acid–based tests are the most common molecular tools within clinical microbiology laboratories. They are versatile, robust, performed rapidly, are highly specific and quite sensitive (especially the amplification tests), and can often be done directly from the clinical specimen, mitigating the need to wait for culture results. They are the standard of care for organ isms that are difficult or impossible to culture (e.g., Bordetella, Chlamydia, and Mycoplasma), and they have largely replaced diagnostic approaches that relied on microscopic observation (e.g., Entamoeba, Cryptosporidium, and Trichomonas).
NAATs use polymerase chain reaction (PCR) or other amplifying process to increase the number of specific DNA or RNA molecules so the sensitivity of the test is significantly higher than that of unamplified tests. Contemporary assays can target a single pathogen or use multiplexed panels now containing up to 30 targets to identify pathogens associated with particular clinical syndromes (e.g., pneumonia, endocarditis, or meningitis). Examples of FDA-approved NAAT tests include C. trachomatis and N. gonorrhoeae in urine samples in sexually transmitted diseases, the Luminex respiratory virus panel for detection of multiple respiratory viruses, and the BioFire Film Array meningitis/encephalitis (ME) panel, which simultaneously detects 14 common infectious agents in CSF. This technology not only detects pathogens but can also be used to detect certain drug-resistance genes. For example, the Xpert MTB/RIF assay detects M. tuberculosis infection in under 2 hours and detects resistance to rifampin.
Tests that use nucleic acid probes are designed to detect DNA or RNA directly (without amplification) using a labeled DNA or RNA probe that hybridizes specifically to the nucleic acid from a cultured organism. These tests are simpler to perform than the amplification tests but are less sensitive.
Nucleic acid sequence analysis of ribosomal RNA (rRNA) can be used to identify bacteria or fungi. This is considered a universal approach because it is based on amplification of genes that are highly conserved within a given organism type, such as the 16S and 23S rRNA genes in bacteria and the 28S rRNA and internal transcribed spacer (ITS) genes in fungi. A bacterium that had never been previously cultured, Tropheryma whipplei, was identified using this approach.
Next-generation sequencing (NGS) is an umbrella term that includes several types of high-throughput sequencing methods where millions of nucleic acid fragments are simultaneously and independently sequenced, assembled, and then analyzed using powerful computational approaches that compare a sample to a reference database for identification of the pathogen. Metage nomic NGS (mNGS) refers to sequencing all nucleic acids in a clinical sample, which includes host nucleic acid and mixed populations of microbes. This powerful platform was initially developed and approved for clinical testing for oncology and inherited disease applications; however, the potential for clinical applications in infectious disease diagnostics is tremendous. At this point, mNGS has been successfully used to identify pathogens from a variety of clinical specimen including blood, CSF, respiratory samples, and gastrointestinal fluid. The major strength of mNGS is that it is an unbiased diagnostic method that does not presume the identity of the causative agent of disease, but broadly detects all nucleic acids in a sample. While there is great promise and excitement about this technique, there are also substantial concerns about validity and reliability of the resulting information regarding the clinical significance of the organism(s) detected. Since many clinical specimens are collected from nonsterile body sites that harbor a normal flora, not to mention true “contaminating organisms,” detecting this flora in an mNGS assay can lead to misidentifying the true pathogen. This is a major problem since the point of these techniques is to inform treatment decision-making and guide patient care. Several clinical labs have developed risk mitigation strategies to help avoid misinterpretation of mNGS results due to contamination, which include using alternative methods to confirm organisms identified by mNGS and implementing strict contamination-monitoring procedures. Currently, this technology is only available on a limited basis from a few select laboratories, including the University of California, San Fran cisco Clinical Microbiology Laboratory, although tests based on this approach are rapidly being developed. Because these types of sequencing approaches only confirm the presence of a nucleic acid target and do not prove the presence of a viable organism, these assays are often performed in concert with other culture dependent methods.
There is great interest in developing molecular point-of-care testing (POCT), including in clinics and urgent care settings. The idea of POCTs is not new; clinics have used POCTs to detect antigens or antibodies for infections such as influenza, mononucleosis, and group A Streptococcus (GAS) (Rapid Antigen Detection Tests [RADT]). However, these assays have lower sensitivity and specificity than their molecular counter parts. For instance, the FDA approved a molecular diagnostic test based on detection of GAS-specific DNA sequences. This NAAT is much more sensitive and specific than the RADT, which can detect <50 bacterial cells/mL of GAS from a throat swab within 15 minutes, but the antigen detection test is still significantly faster than the 24 hours required for plate cultivation. POCT impacts initiation of definitive antimicrobial therapy, can help to prevent unnecessary therapy, and facilitates infection prevention practices, all of which align with institutional efforts on antimicrobial stewardship to improve quality of care. Currently, molecular POCT for several sexually trans mitted infections (Trichomonas, Chlamydia trachomatis, and Neisseria gonorrhoeae) are under development.
Proteomic Tests
More and more clinical labs are incorporating MALDI-TOF technology into their diagnostic tool repertoire, which is the preferred culture-based diagnostic method both from a cost and time-effective perspective. MALDI-TOF technology measures particles based on their mass-to-charge ratio. In this technique, organisms (currently limited to bacteria and some types of fungi) cultured and purified from clinical samples are embedded in a matrix material that, when excited by a laser, transfers charge from the matrix to the microbial macromolecules causing desorption of the newly ionized particles. These charged particles are then separated by their mass-to-charge ratio, yielding a mass spectral signature that is unique to a specific genus and often to the species level. Using bioinformatics, these MALDI-TOF spectra are compared to standardized databases, and those that are highly aligned are identified as a match with a stated level of confidence. This assay, which takes less than a minute to complete once the sample is loaded into the machine, has been shown to be highly accurate, efficient, and cost-effective. Currently, MALDI-TOF cannot accurately differentiate among all species within certain groups of organisms, such as the Enterobacter cloacae complex, Burkholderia cepacia complex, and Streptococcus bovis species group. In addition, Shigella and E. coli cannot be reliably differentiated by MALDI-TOF. Since E. coli is one of the most frequent organisms encountered in clinical microbiology laboratories, alternative strategies must be employed.
There are also several culture-independent diagnostic tests (CIDTs) now available for suspected GI pathogens, which are based on immune assays to detect toxins, or nucleic acid amplification to detect pathogens. These tests are more expensive than culture-based testing, but are highly sensitive and specific, and typically increase detection of all bacterial GI pathogens, especially those that are harder to cultivate (i.e., Campylobacter and Shigella). The main advantages of CIDTs are the speed at which a diagnosis can be obtained (hours versus days for cultivation) and their reliability. CIDTs should be confirmed by culture especially if susceptibility testing is indicated or if there is a suspected outbreak requiring a Public Health investigation.
Emerging Technologies
An exciting emergent diagnostic technology involves the use of clustered regularly interspaced short palindromic repeats (CRISPR). The functional characterization demonstrating that CRISPR-Cas systems are acquired immune systems, widespread in the prokaryotic world, led to the development of innumerable applications using this gene editing tool. This system recognizes foreign nucleic acids in a sequence-specific manner and eliminates them using an endonuclease, now referred to as the CRISPR-associated (Cas) enzyme. CRISPR–Cas systems rely on a special CRISPR RNA (crRNA), which guides Cas enzymes to target sequences and cleaves them. Scientists have designed crRNAs to recognize and hybridize to specific DNA or RNA region of interest, which results in the Cas enzyme cleaving the nucleic acid. This triggers a cellular DNA repair mechanism that can remove or replace a very specific region of nucleic acid. This remarkable finding has been exploited to develop methods that allow for detecting specific nucleic acid sequences with exquisite sensitivity. For instance, SHERLOCK (Specific High sensitivity Enzymatic Reporter unlocking), is a detection plat form that combines nucleic acid amplification of a desired target followed by Cas-mediated cleavage using a reporter RNA that is designed to recognize and hybridize to the desired target. Cas enzyme cleavage of the target nucleic acid produces a detectable signal that indicates the presence of the target sequence. This system is highly specific and sensitive, allowing for the detection of very small amounts of nucleic acids in a sample, making it a powerful tool to detect pathogens. This system has been successfully applied to detect nucleic acids from emerging viral threats, such as Zika virus and dengue virus.
