INFECTION DIAGNOSTICS :: MICROBIOLOGY of colony is needed for this approach.8


A


more mass and widespread approach, metagenomic sequencing, involves se- quencing all available templates within a clinical sample including pathogen and human DNA and RNA. This approach is advantageous in that it does not requiring culturing and takes an unbiased approach, enabling for the detection of numerous pathogens (and the associated host re- sponse against them). Finally, targeted NGS is similar to metagenomic yet is more refined, focusing on a subset of genes. Tar- geted NGS first enriches for sequences of interest before the preparation to enhance analytical sensitivity. Contingent upon the focus of the specific disease or disorder, this can range from several to a few hundreds of gene targets.3,10


For example, panels can


be specialized to target bacteria, viruses, and eukaryotic pathogens.3 Whole genome sequencing’s ability to sequence and assemble an entire genome with plasmids is advantageous within the clinical laboratory as a means to identify antimicrobial resistance profiles thereby influencing first-line drug implementa- tions. Recently, whole-genome sequencing proved beneficial to detect and character- ize the emergence of several patients suf- fering from pneumonia from an unknown cause from Wuhan, China. While the initial sequence was of unknown origin, later bioinformatic analysis identified simi- larity to beta-coronaviruses and termed SARS-CoV-2 thus exemplifying the benefit of whole genome sequencing in identify- ing novel organisms and/or mutations.3,10 Metagenomic NGS has proven benefi- cial when targeted or less comprehensive tests fail. Given its wide inclusivity and lack of requirement of previous knowl- edge of potential pathogens, several clinical tests have been developed from a variety of patient samples including: synovial fluid, CSF, feces, corneal tissue, blood, plasma, nasopharyngeal swabs, and joint fluid as a proxy to diagnose various types of infections.3,10


Body fluid


samples can possess significant complex- ity in terms of the biodiversity present and metagenomic NGS enables for detec- tion of low-prevalence templates within the entire sample that would have likely been missed by other diagnostic means.3 A limitation of Metagenomic NGS is the disproportionate ratio of host to pathogen nucleic acid reads thus decreasing the analytical sensitivity of the assay. Targeted NGS improves analytical sensitivity by first enriching for highly conserved regions of pathogens, such as the 16S rRNA in bac- teria. Targeted NGS has proven beneficial in terms of contributing to public health, such as enriching for SARS-CoV-2 RNA


22 JUNE 2022 MLO-ONLINE.COM


in clinical samples as a means to track the rise of variants.3,10


Targeting NGS of both


the host and its associated flor3a can serve as an indicator of the general well-being of the patient. For example, sequencing of the gene expression of a patient’s immune response gene profile combined with se- quencing of commensals and pathogen genomes lead to the correct identification of the causative agent with high sensitivity and specificity with a true negative predic- tive value of 100%. Likewise, sequencing of the virome within immunocompro- mised patients can serve to evaluate the competency of the host immune system, if viral loads dramatically increase under immunosuppressants. These examples highlight just a few examples of the massive degree of pub- lications available of NGS. It is wide- accepted that NGS possess immense value in contributing to the clinical utility within the healthcare setting. However, these assays are not without flaws. Con- trarily, advances are ever ongoing to help contribute to easier adaption within the clinical lab and better patient outcomes.3 The Current Limitations to Widespread Implementation: It is accurate to claim that NGS sequencing as a diagnostic tool is still in its infancy.10


Currently, the most com-


monly utilized NGS platforms are limited by short reads, reliant upon clonal PCR, have high error rate, requires advanced technical expertise, and guidelines are not universally standardized.3,10


The process


of implementing NGS sequencing re- quires significant resource investment, including test validation, bioinformatics support, data storage, and overcoming insurance cost hurdles.3


Most testing is


current limited to reference laboratories or academic research centers that can afford such upfront resource investment.2,10


It is


reasonable to assume that as the technol- ogy continues to improve to and advance, the threshold for more widespread adap- tion will decline.10


As with any molecular


based diagnostic assay, testing results alone do not guarantee infection and the asymptomatic colonization.4


Conclusion: Within clinical practice NGS possess mass potential, but as it stands today the most optimal, practical utilization appears to be in patient populations where infection is strongly suspected, yet conventional testing is negative.10


The field would sig-


nificantly benefit from a prospective, con- trolled clinical trial evaluating the clinical utility for unbiased pathogen detection from clinical samples. As it stands today, the majority of publications comprise case reports and retrospective studies


comparing the results to the traditional standard of care.10


It is likely only a matter


of time before completion of such types of studies; thus, such research articles would allow for a more convincing argument for clinicians to adapt the application more readily. Likewise, as continual refinements and improvements to the technology continually emerge, NGS can and will be more easily integrated and streamlined within the clinical setting.


REFERENCES:


1. Duan H, Li X, Mei A, et al. The diagnostic value of metagenomic nextgeneration sequencing in infec- tious diseases. BMC Infectious Diseases. 2021;21(1) doi:10.1186/s12879-020-05746-5


2. Patel R. Advances in Testing for Infectious Diseases-Looking Back and Projecting Forward. Clin Chem. Dec 30 2021;68(1):10-15. doi:10.1093/ clinchem/hvab110


3. Zhong Y, Xu F, Wu J, Schubert J, Li MM. Appli- cation of Next Generation Sequencing in Labora- tory Medicine. Annals of Laboratory Medicine. 2021;41(1):25-43. doi:10.3343/alm.2021.41.1.25


4. Curren EJ, Lutgring JD, Kabbani S, et al. Advanc- ing Diagnostic Stewardship for Healthcare-Associ- ated Infections, Antibiotic Resistance, and Sepsis. Clinical Infectious Diseases. 2022;74(4):723-728. doi:10.1093/cid/ciab672


5. Miller S, Chiu C. The Role of Metagenomics and Next-Generation Sequencing in Infectious Disease Diagnosis. Clin Chem. Dec 30 2021;68(1):115-124. doi:10.1093/clinchem/hvab173


6. Ben Khedher M, Ghedira K, Rolain J-M, Ruimy R, Croce O. Application and Challenge of 3rd Generation Sequencing for Clinical Bacterial Studies. Interna- tional Journal of Molecular Sciences. 2022;23(3):1395. doi:10.3390/ijms23031395


7. Eyre DW. Infection prevention and control insights from a decade of pathogen whole-genome sequenc- ing. Journal of Hospital Infection. 2022;122:180-186. doi:10.1016/j.jhin.2022.01.024


8. Datar R, Orenga S, Pogorelcnik R, Rochas O, Simner PJ, van Belkum A. Recent Advances in Rapid Antimicrobial Susceptibility Testing. Clin Chem. Dec 30 2021;68(1):91-98. doi:10.1093/clinchem/hvab207


9. Parker K, Forman J, Bonheyo G, et al. End-User Perspectives on Using Quantitative Real-Time PCR and Genomic Sequencing in the Field. Tropical Medi- cine and Infectious Disease. 2022;7(1):6. doi:10.3390/ tropicalmed7010006


10. Huanyu Wang SJ. Next-Generation Sequenc- ing for Infectious Diseases Diagnostics. Journel Article. Clinical Laboratory News. September 2021 2021;47(7):10-18.


11. Bani Baker Q, Hammad M, Al-Rashdan W, Jararweh Y, Al-Smadi M, Al-Zinati M. Comprehensive comparison of cloud-based NGS data analysis and alignment tools. Informatics in Medicine Unlocked. 2020;18:100296. doi:10.1016/j.imu.2020.100296


Stephen Vella serves as a Medical Science Liaison for bioMérieux US Medical Affairs Division. His background is in Microbiology and has been working in diagnostics for approximately two years. His primary


role serves to assist as neutral entity mediating scientific dialogue exchange for bioMérieux’s molecular and microbiology diagnostic portfolio


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