A study conducted at Taipei Veterans General Hospital showed metagenomic next-generation sequencing (mNGS) provided an efficient and accurate alternative to blood culture in identifying pathogens in patients with suspected sepsis. The researchers employed a workflow using a fractionation filter to remove host cells and enrich microorganisms, increasing sensitivity and lowering cost, according to the study preprint published on MedRxiv.
Sepsis is traditionally diagnosed with blood culture, and pathogens are subsequently identified with additional assays. However, blood culture suffers several shortcomings: culture does not detect viruses or parasites, some bacteria are difficult to culture, culturing can have a long turnaround time, and mixed infections can be difficult to identify. To overcome these limitations, researchers at Taipei Veterans General Hospital compared a novel host depletion filter from Micronbrane Medical, followed by an efficient mNGS workflow against mNGS without a filter and traditional blood cultures.
The study showed Micronbrane Medical’s Devin Fractionation Membrane enabled a genomic DNA (gDNA) based workflow, the Pathogen Real-Time Identification by Sequencing (PaRTI-Seq), achieved higher positivity rates (82 percent) compared to mNGS without a filter (56 percent) or blood cultures (24 percent) and delivered results within 24 hours.
The Pressure on Current Diagnostic Modalities
The need for rapid and accurate infectious disease testing is growing due to the pace of emerging pathogens and global infectious disease events. Current diagnostic modalities, including cultures, PCR, and serology, each have limitations including the inability to detect mixed infections, long turnaround times, low diagnostic yields and cost.
MetagenomicNGS is gaining popularity because it can provide faster and precise identification of all types of known and unknown pathogens. Yet, mNGS is not widely used due to various reasons including cost and sensitivity. One of the major challenges of mNGS in human specimens is host interference, which leads to lower sensitivity of pathogen detection interfered by huge human background and high sequencing costs.
Increasing the Sensitivity of mNGS
Without any form of host filtering, only a small proportion of sequencing reads correspond to potential pathogens. According to Micronbrane Medical, their patented Devin Zwitterionic Interface Ultra-Self-assemble Coating (ZISC) technology removes greater than 99 percent of host nucleated cells from up to 10 mL of biological fluid in just five minutes. Zwitterionic networks have higher stability in complex mediums and provide strong hydration to the membrane, which acts as an energetic barrier against adsorption and biofouling by proteins, bacteria, and blood cells. Micronbrane Medical’s modified ZISC Technology filter effectively reduces leukocytes and removes host contamination from the sample.
By depleting host nucleated cells, microbial DNA is enriched. In fact, the research found that the Devin filter, enabled a genomic DNA (gDNA) based workflow, the Pathogen Real-Time Identification by Sequencing (PaRTI-Seq), increased reads per million (RPM) over 100-fold compared to mNGS without a filter.
Decreasing the Cost of mNGS
Depleting host DNA is a major contributor to total cost reduction in mNGS pipelines. This is because genomes of prokaryotic pathogens are smaller relative to the animal or human genome. However, for mNGS to replace culture, PCR, or serology techniques in molecular laboratories, all aspects of the NGS process — sample preparation, DNA/RNA extraction, library preparation, sequencing, and data analysis — must be optimized.
The PaRTI-Seq workflow starts with the Devin Microbial DNA Enrichment Kit. The Devin filter effectively removes human nucleated cells from whole blood, plasma, cerebrospinal, bronchoalveolar lavage, vitreous humor or other fluids within five minutes, while ensuring a high microbial passing efficiency due to its large pore size (15 to 20 µm). The Devin filter ensures effective mitigation of host DNA contamination and allows the enrichment of high-molecular-weight microbial DNA. To accommodate variations within research and clinical laboratories, the Devin Enrichment Kit includes manual and automatic protocols, making it compatible with multiple downstream applications including NGS on all platforms.
By effectively reducing host contamination, the sequencing output required to achieve a representative microbiome sample can be reduced five times. This reduces sequencing costs or shortens the sequencing time, depending on the downstream sequencing platforms.
The next step in the PaRTI-Seq assay is to ensure that the library preparation protocols are miniaturized for pathogen detection. Micronbrane Medical’s Unison Ultralow DNA NGS Library Preparation Kit is a workflow that accelerates turnaround time in addition to streamlining costs. In about 2.5 hours, Unison reduces sequencing repetitions and shortens sequencing preparation time.
Innovating Metagenomics
The Devin filter and PaRTI-Seq assay streamlined the sample-to-result processes, reducing the overall turnaround time and cost per sample. Moreover, the gDNA-based PaRTI-Seq assay performed even better than cell-free DNA-based mNGS, showing an average reads-per-million (RPM) of 2,359 compared to only 95 by a cfDNA-based method. The study concluded that mNGS with the Devin filter was able to recover most of the pathogens identified by clinical blood culture and achieved the highest diagnostic yield. With the clinical implementation to complete the workflow within 24 hours, it has the potential to overcome slow turnaround and low diagnostic yield issues of traditional blood culture.
Micronbrane Medical say they hope the Devin filter and PaRTI-Seq assay will accelerate widespread adoption of mNGS by enabling fast and accurate identification and monitoring of a broad range of microorganisms, including bacteria, fungi, viruses, and parasites in both research and clinical settings. Ultimately, they say, the goal is to provide mNGS insight to transform our relationship with the microbial world to benefit health and sustainability.
To learn more, visit https://micronbrane.com/tvghstudy1/.