Small-Volume Endophthalmitis mNGS App Note
Endophthalmitis is a sight-threatening intraocular infection where the diagnostic sample is often small, precious, and difficult to recollect. The Micronbrane small-volume app note shows how the PaRTI-Seq workflow was adapted for a 50 uL vitreous humor sample from a patient with endophthalmitis, then used to identify the predominant pathogen, Klebsiella pneumoniae.
This resource summarizes the workflow and key findings from the app note. It is intended for clinical microbiology researchers, laboratory teams, and mNGS workflow evaluators working with low-volume sterile body fluid samples.
Why Small-Volume Eye Samples Are Difficult for mNGS
Culture-based methods can take days, and negative cultures are reported in a substantial share of endophthalmitis cases despite the disease being microbial in origin. Vitreous humor also creates a practical mNGS challenge: sample volume can be extremely limited, and host-derived nucleated cells can dominate sequencing reads before the microbial signal becomes visible.
For small-volume specimens, the workflow must preserve as much microbial material as possible while reducing the host background that consumes sequencing capacity. That is the role of the Devin Host Depletion Filter in the PaRTI-Seq workflow.
Workflow Used in the App Note
The app note describes a modified small-volume protocol for vitreous humor and eye-swab samples. A 50 uL vitreous humor sample was diluted to 500 uL with sterile phosphate-buffered saline. A swab from the uninfected eye was immersed in 500 uL PBS and processed as a negative comparator.
The samples were passed through the Devin filter, then washed with additional PBS to collect roughly 1 mL of filtrate. After centrifugation, DNA was purified from the pellet and converted into sequencing libraries with the Unison Ultralow DNA NGS Library Preparation Kit.
Sequencing generated 100 bp reads, which were analyzed with Micronbrane’s PaRTI-Cular bioinformatics pipeline. The broader workflow connects sample processing, low-input library construction, sequencing, and PaRTI-Seq Analysis into a sample-to-result mNGS process.
Key Findings
The PaRTI-Seq workflow identified Klebsiella pneumoniae as the predominant pathogen in the vitreous humor sample. This result agreed with culture data, providing an mNGS-based confirmation from a small-volume specimen where preserving sample material is critical.
The analysis compared microbial relative abundance in the vitreous humor sample against the uninfected-eye comparator. Klebsiella species showed relative-abundance ratios above the signal background, with K. pneumoniae standing out as the primary organism. Other Klebsiella signals may reflect conserved sequences or partial genome coverage, so the app note treats K. pneumoniae as the main supported call.
The result matters because it demonstrates that a host-depletion and ultralow-input library-prep workflow can recover actionable microbial signal from a very small ocular sample.
What This Means for Low-Volume Infectious Disease Samples
The app note focuses on endophthalmitis, but the workflow problem is broader. Many clinically important samples are hard to recollect or available only in limited volume, including cerebrospinal fluid, synovial fluid, and other sterile body fluids. In these settings, splitting the sample across many targeted tests can exhaust the specimen before the causative organism is found.
An unbiased mNGS workflow can help when the differential diagnosis spans bacterial, fungal, viral, or parasitic causes. For additional sterile body fluid validation context, see mNGS Validation for Sterile Body Fluids: Methods and Results.
Download the App Note
Download the Small-Volume Endophthalmitis App Note (PDF)
Products and workflow components referenced in this app note:
- Devin Host Depletion Filters
- Devin Microbial DNA Enrichment Kits
- Unison Ultralow DNA NGS Library Preparation Kit
- PaRTI-Seq Analysis
