Advances in molecular diagnostics like multiplexed PCR, waived point-of-care NAAT, and next-generation sequencing (NGS) are quickly becoming overshadowed by newer technologies. A new era of sophisticated biomarker testing and rapid antimicrobial susceptibility testing (AST) is demanding the attention of all clinical microbiology laboratories.
We are seeing the field of sepsis diagnostics roll-out new tech targeting host immune response and biomarker measurements. In AST, the realm of rapid phenotypic testing is evolving through measuring volatile organic compounds, measuring morphokinetic changes, or performing rapid microbroth dilution.
Yet the foundational techniques of broad-range polymerase chain reaction (BR-PCR) and genomic sequencing continue to make strides toward clinical relevance and adoption. This is partially due to lower cost barriers but also increasing end-user experience and clinical need.
While newer technologies offer tantalizing prospects for disease detection, the robustness, versatility, and ever-expanding applications of BR-PCR and genomic sequencing ensure its continued relevance within clinical microbiology.
What is broad-range PCR?
BR-PCR is a molecular technique that requires the presence of microbial nucleic acid in a clinical sample. Bacteria, like other organisms, have ribosomes composed of RNA and protein. The 16S rRNA gene, which encodes a structural component of the ribosome, contains highly conserved regions that are universally present in bacteria, making it a useful marker for identifying them. Similarly, fungi can be identified by targeting the internal transcribed spacer (ITS) regions or the 28S rRNA gene.
Targeting a highly conserved region allows for the design of universal primers. During BR-PCR, primers bind to the conserved target region and amplify both the conserved and adjacent hypervariable regions, which are organism-specific. The resulting material can then be submitted for NGS, where the conserved and hypervariable regions can be used to identify the organism.
BR-PCR for infectious disease is performed on numerous sample types, including but not limited to, sterile tissue, paraffin-embedded tissue, sterile body fluids like blood, lower respiratory tract specimens, and pure bacterial colonies.
What is targeted next-generation sequencing (tNGS)?
NGS takes a broadly agnostic approach toward sequencing all nucleic acids directly from a patient sample. This unbiased approach enables detection of microbes, as well as resistance genes and traits. NGS can also be utilized as follow-up to BR-PCR for a more selective detection and identification approach. Whether targeting the 16S rRNA gene for bacteria or the ITS region for fungi, this selective approach can be referred to as targeted NGS (tNGS).
Following BR-PCR, the material of conserved and hypervariable regions are submitted for massively parallel sequencing of the DNA and/or RNA. The generated sequence reads are compared against comprehensive public databases (e.g., GenBank, RDP, and SILVA) to identify the bacterial genera and species present in the sample.
This powerful approach enables the identification of uncultivable or fastidious microorganisms that are difficult or impossible to detect by traditional methods. Potential uses include the diagnosis of culture-negative infections, identification of novel pathogens, characterization of polymicrobial infections, and investigation of microbial communities in various body sites, such as the gut microbiome or chronic wound infections.
Clinical applications of targeted NGS
In situations where traditional methods fail but an etiology of infectious disease is strongly suspected, tNGS has potential to improve the standard of care. Use cases for tNGS include prosthetic joint infection (PJI), central nervous system (CNS) infections, or suspected bacteremia—i.e., bacteria in the bloodstream.
Culture negative endocarditis (an infection of the inner lining of the heart) is a prime opportunity for tNGS as traditional blood cultures often struggle to detect etiology, requiring multiple sets of blood cultures to reduce false negative findings. Unlike PJI and CNS infections, no current FDA approved multiplex PCR option exists as a supplementary test for culture negative specimens. In cases of suspected bacteremia, NGS may be performed on EDTA whole blood or plasma upon initial patient presentation or as conventional testing comes up negative.
Detecting tickborne pathogens with tNGS
Tickborne disease is a particular area of interest for tNGS as current diagnostics for acute disease include low sensitivity methodologies like serology or peripheral blood film review. Etiology-specific nucleic amplification tests are improving diagnosis in the acute phase but only when a detailed patient history is available, as clinical presentations are often vague. Additionally, some laboratories offer these as individual assays, allowing for target misses and leading to prolonged times to diagnosis. As the endemic regions of tickborne diseases expand and new etiologies like Alpha-gal syndrome are discovered, relegating tickborne testing to a select few etiologies may prove clinically unsound. Unlike tNGS, unbiased metagenomic sequencing can be used to detect emerging tickborne pathogens.
Limitations of targeted NGS
Despite its undeniable strengths, tNGS testing is not without its limitations and drawbacks. One significant challenge is the clinical interpretation of tNGS findings.
As it detects DNA from both living and dead bacteria, this can complicate interpretation in patients who have previously received antibiotic treatment.
Clinical interpretation is further complicated by polymicrobial infections and contamination risks. Bacterial DNA is ubiquitous in the environment and can even be found among laboratory collection supplies and reagents. Determining true polymicrobial infections from those that are contaminated during the collection and testing process requires significant expertise. Incorrect diagnosis or unnecessary changes in treatment can negatively affect patient outcomes.
The very nature of BR-PCR and sequencing being targeted to specific bacterial or fungal genes can contribute to misses for other infections. Therefore, some number of conventional tests like serology and culture are still required in many investigations. This may be overcome by use of metagenomic sequencing instead of tNGS but at significant time and cost.
Another limitation is its relative inability to provide information on antimicrobial susceptibility, a critical component of infection management. Although advances are being made to infer susceptibility from genomic data, traditional culture and phenotypic susceptibility testing remain the gold standard for guiding antibiotic therapy.
Cost is another important limitation of tNGS testing, as combining 16S PCR and NGS is significantly more expensive than routine diagnostic tests. Therefore, these tests warrant careful consideration of their clinical impact and integration into routine clinical workflows. Failure to do so may lead to overutilization and wasteful spending, compounded by unclear clinical interpretation and further diagnostic testing.
Insurance coverage poses another barrier to widespread adoption of tNGS in routine clinical practice. Inconsistent reimbursement policies across insurers discourage laboratories from adopting this test into routine workflows. Insurance coverage decisions are often based on studies showing that test results can change clinical decision-making or improve patient outcomes. With difficult to meet requirements, laboratories will delay onboarding new assays if it risks increasing operational costs or places the financial burden singularly on patients.
Finally, there have also been reports of limited clinical impact in the literature, with one study that covered multiple specimen types showing tNGS changed clinical care in only 6% of cases.
Best practice guidance for targeted NGS
Societal guideline recommendations for the use of NGS in infectious disease remain limited. The most current Infectious Diseases Society of America (IDSA) and American Society for Microbiology (ASM) guidance relegates the use of targeted sequencing for use after common etiologies have been ruled out. This is problematic because it defers utilization and coverage decisions to insurance companies. As noted previously, this limits broader adoption by making testing appropriateness contingent on insurer approval.
Appropriate implementation of NGS has been shown to have significant clinical impact in multiple studies across various specimens and indications. One retrospective study of unfiltered use of tNGS suggests that culture negative infections of patients who recently received antibiotic pretreatment can yield clinically valuable information. Further, use of tNGS and its clinical impact can vary greatly by specimen and infection type (blood, synovium, respiratory, tissue, etc.).
The literature consistently highlights that the value of tNGS depends on multiple factors. These factors include the timing of testing within diagnostic workup, assay turnaround time, the potential for therapy change, and the relevance of findings to the patient’s clinical context. An additional consideration is whether the specimen contains a fastidious organism that cannot grow in conventional culture.
Detecting fastidious organisms with tNGS
At Emplify Health, we have seen the most benefit of tNGS for identifying fastidious organisms that are present on Gram stain but fail to grow on traditional media, particularly in sterile samples like tissue and blood cultures, where fastidious organisms are often found.
In our workflow, culture begins in our laboratory, but if culture growth is negative, the sample may be sent to our referral laboratory for BR-PCR and sequencing. In one case, a pleural fluid with moderate gram-negative rods on Gram stain failed to grow in traditional culture. This prompted our microbiology team to pursue tNGS on the pleural fluid, which detected Capnocytophaga canimorsus, slow-growing bacteria that are difficult to culture in the lab.
In these select culture-negative cases, tNGS has been useful for detecting pathogens within a controlled utilization format and guided potential treatment changes for patients. While tNGS can be ordered by any provider in our system, it is used selectively and is typically initiated only when an infectious disease specialist is consulted and recommends it for difficult cases.
The future of targeted NGS
As novel biomarkers and rapid AST gain attention, it is important to recognize the continued development and increasing value of tNGS as a key diagnostic tool for microbial identification.
tNGS is steadily finding its place in clinical and laboratory settings, but continued growth depends on its routine adoption. In the absence of clear industry guidance, laboratories must develop internal workflows, as well as experiment with NGS to demonstrate its clinical utility and value in specific cases. As laboratories gain experience in performing and interpreting these tests, and as practice recommendations evolve into standardized workflows and algorithms, the utility of tNGS will grow. Ongoing advances in broad-range target genes, new sequencing technologies, and improved bioinformatics tools for microbial community analysis are further refining the technique.
The clinical community increasingly recognizes the value of tNGS for infection detection as the limitations of culture-based diagnostics become more apparent in certain diseases and in pathogen discovery. tNGS will remain an indispensable tool, complementing conventional technologies and offering a robust, nuanced perspective on microbial involvement in a vast array of clinical conditions.
