Differential lysis depletion methods – drawbacks, shortcomings and alternatives

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Detection of pathogenic DNA from blood samples is essential in diagnosing bacterial, fungal, and viral diseases. Still, the amount of genetic information found in analyzed samples is extraordinary. Interference from host DNA decreases sensitivity for microbial detection and represents a significant drawback in identifying the pathogenic microorganism1.

 

What are the current lysis depletion methods, and how do they work? 

Chemical or enzymatic techniques

These methods imply using a lysis solution such as a saponin and DNase treatment for the host DNA depletion. Chemical and enzymatic techniques decrease up to 99.9% or 105 folds of the human DNA in the samples to be analyzed. Saponin depletion can effectively remove the human genomes while preserving most pathogenic bacterial genomes. However, the pathogenic genome does not remain completely unaltered. This method requires 5–6 h from sample collection to pathogen classification.

Osmotic lysis

In this method, the lysis process is followed by propidium monoazide (PMA) or benzoate treatment to reduce host reads and increase microbial reads in the analyzed samples. Currently available lyse kits declare a host read decrease as low as 8 to even 5%. The required time just for lysis ranges from 40 to 100 minutes. One of the main advantages of osmotic lysis and PMA treatment methods is that this method requires fewer steps than other enzymatic methods and is also more cost-efficient2, 3.

Devin® filter

Devin® filter uses bio-compatible membranes to separate blood components and selectively remove up to 99.99% of white blood cells from the filtered sample. Devin filters do not interfere with the bacterial, viral, and fungal communities during the filtration process. As such, the pathogenic agents responsible for infections can be detected in the processed sample through nucleic acid isolation and amplification techniques. For testing, blood samples obtained from healthy donors were enriched with 10^4 Genome Copies/mL spike-in control (ZYMO Research) and then run through the Devin® filter. Results showed a quicker processing time and better enrichment results when compared to traditional methods4.

What are the limitations of currently used depletion methods, and what are the alternatives?

Bacterial cell wall structure variates from one species to another and significantly influences the lysis process. Consequently, the obtained results can be distorted by the bacterial cell lysis efficiency, and the relative abundance of bacteria in the analyzed sample can be misread. Studies developed on standardized microbial DNA samples revealed that the methods currently used to extract bacterial DNA can yield significantly different results.

The techniques observed to recover the lowest bacterial DNA used high temperature, ASL buffering solution, and protein kinase K for the lysis process. On the other hand, the highest microbial DNA yield was obtained on a method that used phenol-chloroform-isoamyl alcohol for extraction on which bacterial recuperation was  5.7, 5.4, and 3.3-fold higher on average for bacteria such as S. aureus, Pr. acnes, and C. tuberculostearicum. Saponin depletion methods can affect some microorganisms, such as Streptococcus pneumoniae, an important pathogen that can cause severe disease in humans5 6. Further, in the osmotic lysis, denaturation and disruption induced by PMA in the structure of proteins and nucleic acids can also affect extracellular bacterial DNA, lyse the bacteria and reduce the pathogenic community. Another critical limiting factor is the removal of intracellular viral material. Also, the high number of steps required to process a sample can affect the viability of the pathogenic community2, 3.

Devin® filter is among the best alternatives to currently used methods. The main advantage is that the technique does not require the usage of any chemical compound or lysing agent. Devin filters deplete blood samples of 99.99% of WBC and allow the unaltered passing of microorganisms. Compared to other tests, the depletion method of Devin filters is ideal for microbial enrichment of whole blood and different types of body fluids that require further metagenomic tests4.

Depletion techniques provide a new approach for rapidly identifying pathogens in clinical microbiology. Depletion methods have a higher sensitivity and require less time to establish a correct diagnosis than a clinical culture. Although highly reliable, depletion methods still have a series of critical shortcomings in depleting host genetic information, preserving pathogenic DNA, and ruling out a pathogenic microorganism as a causal agent for an infection. Techniques such used in Devin® filter overcome these limitations and could be key to cell depletion and clinical microbiology.

Reference

  1. Heravi, F. S., Zakrzewski, M., Vickery, K., & Hu, H. (2020). Host DNA depletion efficiency of microbiome DNA enrichment methods in infected tissue samples. Journal of Microbiological Methods, 170, 105856. https://doi.org/10.1016/j.mimet.2020.105856
  2. Charalampous, T., Richardson, H., Kay, G. L., Baldan, R., Jeanes, C., Rae, D., Grundy, S., Turner, D. J., Wain, J., Leggett, R. M., Livermore, D. M., & O’Grady, J. (2018). Rapid Diagnosis of Lower Respiratory Infection using Nanopore-based Clinical Metagenomics. Rapid Diagnosis of Lower Respiratory Infection Using Nanopore-Based Clinical Metagenomics. https://doi.org/10.1101/387548
  3. Hasan, M. R., Rawat, A., Tang, P., Jithesh, P. V., Thomas, E., Tan, R., & Tilley, P. (2016). Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing. Journal of Clinical Microbiology, 54(4), 919–927. https://journals.asm.org/doi/10.1128/JCM.03050-15
  4. Micronbrane (2021, November 17). [White Paper] Needle in the Haystack: How to Remove Human Background When You Want to Detect Microorganisms – micronbrane. Micronbrane – Micronbrane.Com. https://micronbrane.com/white-paper-needle-in-the-haystack-how-to-remove-human-background-when-you-want-to-detect-microorganisms/
  5. Wu, N., Ranjan, P., Tao, C., Liu, C., Yang, E., He, B., Erb-Downward, J. R., Bo, S., Zheng, J., Guo, C., Liu, B., Sun, L., Yan, W., Wang, M., Wang, W., Wen, J., Yang, P., Yang, L., Tian, Q. Shen, N. (2021). Rapid identification of pathogens associated with VAP by Nanopore sequencing. Respiratory Research, 22(1). https://doi.org/10.1186/s12931-021-01909-3
  6. Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., & Forney, L. J. (2012). Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome. PLoS ONE, 7(3), e33865. https://doi.org/10.1371/journal.pone.0033865

 

 

 

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