What is NGS-based detection in clinical microbiology?
The current gold-standard in clinical microbiology for bacterial, viral, and fungal detection is culture and biochemical testing, referred to here as classic methods, which are widely available in microbiology laboratories. Recently, next-generation sequencing (NGS) has penetrated the clinical microbiology market offering a new approach to detect potentially pathogenic microorganisms.
NGS uses the sequence of 16S rRNA, a highly conserved region in bacterial and fungal genomes to identify microbes within a sample. A sample being submitted for NGS-based microbial detection undergoes a process of lysing cells within the sample, purifying the nucleic acids released during lysis, enriching the sample for 16S rRNA, and if necessary, amplifying the DNA within the sample. Following sequencing, the 16S rRNA sequences are aligned against known microbial 16S rRNA sequences to determine what microbes are present within the sample.
Similar to classic methods, NGS can be used with multiple sample types, such as isolated bacterial colonies, swabs, aspirates, blood samples, etc. As NGS detection relies on amplified DNA, there is no requirement to culture or isolate individual bacterial colonies to identify the microorganisms present in a sample, and it can present a more complete picture of the microbiome within that sample.
What are some of the shortcomings of classic microbe detection methods?
The classic microbe detection methods are still considered the gold-standard; however, these techniques have shortcomings that other technologies, like NGS, can address. The first being that not all microbes, such as fastidious bacteria and anaerobic bacteria, can be cultured under standard laboratory conditions.
Fastidious microbes known to cause human disease include viruses, Borrelia burgdorferi (Lyme disease), Treponema pallidum (syphilis), and Mycobacterium tuberculosis (tuberculosis). While pathogenic obligate anaerobic bacteria include Clostridium botulinum (botulism), Fusobacterium necrophorum (Lemierre syndrome), and Campylobacter jejuni (gastroenteritis).
This shortcoming also extends beyond bacteria, as viruses and fungi can also be difficult, if not impossible, to detect using standard culture practices. This is where NGS-based detection can provide a significant advantage to classic methods as it does not require culturing or isolating microbes prior to identification, and can identify antibiotic resistance genes without additional culturing of bacteria for sensitivity testing.
A final shortcoming of classic microbe detection is that pathogenic viruses cannot be detected using standard culture and biochemical profiling, which can result in false negative results for patients with viral infections. Although NGS cannot be used to detect RNA viruses without modifying the protocol, NGS can detect pathogenic DNA viruses, including the varicella-zoster virus (chicken pox/shingles), Ebstein-Barr virus (mononucleosis), and HIV (AIDS). Thus, NGS can be used as an unbiased culture-independent method to detect pathogenic viruses.
What are the limitations of NGS-based microbe detection?
The limitations of NGS in clinical microbiology include unclear antibiotic resistance, inability to detect which pathogen is causing disease, costs, and unknown clinical utility.
NGS is capable of detecting many known antibiotic resistance genes, but it is important to note that not all resistance genes are known or highly conserved among pathogens. For example, a retrospective study investigating antibiotic resistance in Klebsiella pneumoniae found that the NGS assay had an accuracy of 92 percent when measuring the minimum inhibitory concentration of 20 antibiotics relative to classic techniques. This finding indicates that nearly 9 percent of cases are at risk of receiving improper treatment due to this discrepancy. Similar accuracies were reported in Pseudomonas aeruginosa and Staphylococcus aureus.
Furthermore, NGS technology is capable of reading fragments that are smaller than a single gene and assembles those fragments into a genome, which means it is impossible to determine which organism a resistance gene originated from in a mixed sample such as uncultured wound or aspirate samples. Although clinicians may decide to treat the entire sample as though it is resistant, this could lead to the overuse of antibiotics.
Another limitation for NGS in clinical microbiology is the inability to determine the causal microbe in a mixed sample. A typical example of this situation is healthy individuals identified as methicillin-resistant Staphylococcus aureus (MRSA) carriers, which may be as high as 15.3 percent of health care students. Detection of MRSA via an NGS test could present difficulties and may result in falsely identifying MRSA as the pathogenic microbe in patients carrying MRSA as part of their healthy microbiome. Moreover, in a sample that contains multiple pathogenic microbes, it can be more difficult to identify causal microbes without observing the relative concentration in a classic culture method.
The cost of implementing NGS in clinical microbiology labs is another concern. In 2018, the cost was estimated to be approximately $130 USD per sample (€115 EUR), but that does not include the upfront costs or training prior to implementation. Thus, each lab must consider this cost relative to their cost per sample using a classic detection technique.
With the potentially higher cost, it is important to also consider the clinical utility of NGS. Although there have been many published case reports indicating the importance of NGS in diagnosing microbial infections that would have otherwise been missed by classic methods, the clinical utility of NGS in clinical microbiology is not yet proven, as illustrated by a recent publication in the Journal of Clinical Microbiology showing limited clinical utility in a cohort of 80 patients with potential cerebral spinal fluid infections. The authors concluded that positive results were often of unclear clinical significance, however the sample size was small for this type of analysis.
Weigh the pros and cons before adopting NGS for microbe detection
Using NGS in the clinical microbiology laboratory may provide benefits via culture-independent identification of pathogens, including fastidious organisms. However, there remain significant questions surrounding the clinical utility and cost of NGS, as well as how an NGS workflow fits into a classic microbiology workflow. Thus, clinical leaders should carefully consider the pros and cons of NGS-based detection prior to adopting it in their labs.