Cited by 6Wound Healing and TreatmentsBacterial biofilms and quorum sensingCell Image Analysis Techniques

The DOI matches its Crossref record; 75 of the 75 references it cites were found in a registry or an index; none of the 98 sources its claims cite is checked for support yet.

SEMTWIST Quantification of Biofilm Infection in Human Chronic Wound Using Scanning Electron Microscopy and Machine Learning

Open access
Singh, Surabhi, Muniz De Oliveira, Fabio, Wang, Cong, Kumar, Manoj, Xuan, Yi, DeMazumder, Deeptankar, Sen, Chandan K., Roy, Sashwati·2025·Advances in Wound Care

Abstract

Objective:To develop scanning electron microscopy-based Trainable Weka (Waikato Environment for Knowledge Analysis) Intelligent Segmentation Technology (SEMTWIST), an open-source software tool, for structural detection and rigorous quantification of wound biofilm aggregates in complex human wound tissue matrix. Approach:SEMTWIST model was standardized to quantify biofilm infection (BFI) abundance in 240 distinct SEM images from 60 human chronic wound-edge biospecimens (four technical replicates of each specimen). Results from SEMTWIST were compared against human expert assessments and the gold standard for molecular BFI detection, that is, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Results:Correlation and Bland–Altman plot demonstrated a robust correlation (r = 0.82, p < 0.01), with a mean bias of 1.25, and 95% limit of agreement ranging from −43.40 to 47.11, between SEMTWIST result and the average scores assigned by trained human experts. While interexpert variability highlighted potential bias in manual assessments, SEMTWIST provided consistent results. Bacterial culture detected infection but not biofilm aggregates. Whereas the wheat germ agglutinin staining exhibited nonspecific staining of host tissue components and failed to provide a specific identification of BFI. The molecular identification of biofilm aggregates using PNA-FISH was comparable with SEMTWIST, highlighting the robustness of the developed approach. Innovation:This study introduces a novel approach “SEMTWIST” for in-depth analysis and precise differentiation of biofilm aggregates from host tissue elements, enabling accurate quantification of BFI in chronic wound SEM images. Conclusion:Open-source SEMTWIST offers a reliable and robust framework for standardized quantification of BFI burden in human chronic wound-edge tissues, supporting clinical diagnosis and guiding treatment.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

SEMTWIST is an open-source, Trainable Weka-based machine-learning tool for segmenting and quantifying biofilm infection (BFI) in scanning electron microscopy images of human chronic wound-edge tissue. It showed strong correlation with expert human ratings and molecular PNA-FISH detection, while culture and WGA staining were less reliable for biofilm-specific assessment.

Key findings

  • SEMTWIST BFI quantification correlated strongly with average trained human expert scores: r = 0.82, p < 0.01, with mean bias 1.25 and 95% limits of agreement from −43.40 to 47.11.
  • Human expert raters showed substantial interrater variability: mean bias 17.8 and wide limits of agreement from −33.34 to 68.93, indicating low reliability/reproducibility of manual BFI assessment.
  • Among 15 patient samples, 8 were culture-negative; 5 of those 8 culture-negative biospecimens showed dense biofilm structures by SEM, and PNA-FISH detected polymicrobial BFI-like structures in culture-negative samples.
  • WGA staining was nonspecific in human chronic wound tissue and showed only weak correlation with TWIST BFI quantification (r = 0.24). Image-resolution sensitivity testing showed BFI abundances of 42.6%, 26.4%, and 25.8% at 320 × 256, 960 × 768, and 2,560 × 2,048 pixels, respectively.

Methods

  • Blinded subset of 240 SEM images from 60 human chronic wound-edge biospecimens, with four technical replicates per specimen; 160 SEM images were used to train the software for BFI and non-BFI features.
  • SEM images were processed with TWIST using a fast random forest classifier and feature filters including Gaussian blur, Sobel, Hessian, difference of Gaussians, membrane projections, variance, mean, minimum, maximum, and median; postprocessing used histogram thresholding and k-means clustering to calculate percentage biofilm coverage.
  • Validation compared SEMTWIST against four blinded human biofilm experts rating 60 SEM images from 15 subjects, PNA-FISH for S. aureus and P. aeruginosa, WGA-Alexa Fluor 488 staining, and standard bacterial culture; analysis used correlation, regression, and Bland–Altman plots.

Limitations

  • Paper-stated: human expert assessment is subjective, time-intensive, limited by expert availability, and prone to interobserver variability; the study observed interrater variability and concluded human rating is valid only when performed by >3 raters.
  • Paper-stated: TWIST performance decreased at lower pixel densities, and low-resolution SEM images with rare (<50%) biofilm may be unreliable; high-resolution images require more processing time and power.
  • Evident from the text: the paper has no dedicated limitations section, and the SEMTWIST validation used a blinded subset from an ongoing, unreported clinical trial whose primary outcomes are not reported here.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1089/wound.2024.0291

Cites

75 of 75 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

40 Check pending · 27 Own result · 21 Own interpretation · 10 No basis given · 9 Own method · 7 No full text of the sources · 3 Not checked yet · 1 Sources not found

Verify all 74 references — free account
  • Probably not a substantial claim. 39
  • It may state two separate claims as one. 28
  • The quote only partly states this claim. 1
65 of 65
  • The paper relies on this without support.

    Wound biofilm is a polymicrobial aggregate including host cells.

    “SEM analysis reveals that wound biofilm, shaped by an iterative exchange between microbial and host-defense processes, is a polymicrobial aggregate including host cells.”
  • The paper relies on this without support.

    Wound biofilm aggregates in chronic human wounds exhibit unique characteristics, and their quantification in clinical samples has long been challenging due to limited understanding of complex structural interactions between biofilm and wound tissue.

    “Wound biofilm aggregates in chronic human wounds exhibit unique characteristics, and their quantification in clinical samples has long been challenging due to limited understanding of the complex structural interactions between biofilm and the wound tissue.”
  • The paper relies on this without support.

    Gradation of BFI abundance is essential to diagnose BFI promptly, allowing for implementation of effective treatment strategies and significantly reducing risk of poor outcomes.

    “This gradation of BFI abundance is essential to diagnose BFI promptly, allowing for the implementation of effective treatment strategies and significantly reducing the risk of poor outcomes.”
  • The paper relies on this without support.

    A reliable quantification approach is essential for clinical research studies evaluating antibiofilm treatments, where BFI reduction is a primary outcome.

    “A reliable quantification approach is also essential for clinical research studies evaluating antibiofilm treatments, where BFI reduction is a primary outcome.”
  • The paper relies on this without support.

    One of the challenges of SEM-based BFI detection in wound-edge tissue is the lack of availability of robust and objective quantification methods.

    “One of the challenges of SEM-based BFI detection in wound-edge tissue is the lack of availability of robust and objective quantification methods.”
  • The paper relies on this without support.

    Variability in human expert assessments often stems from differences in expertise, image noise, and complex anatomical architecture of tissues.

    “This variability often stems from differences in expertise, image noise, and the complex anatomical architecture of tissues.”
  • The paper relies on this without support.

    Biofilm may be embedded within or interspersed between host tissue structures in wound-edge tissue.

    “Given that the biofilm may be embedded within, or interspersed between these structures, accurate segmenting for these structures is necessary.”
  • The paper relies on this without support.

    Human scores are valuable as a gold standard for biofilm quantification.

    “Therefore, while human scores are valuable as a gold standard, it is essential to account for potential biases and discrepancies that may arise from human evaluations.67-69”
8 of 8

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

2 of 2

Citations

Cites

Show all 75 references

Research works printed but not linked: 7

  1. 10.1128/cmr · DOI
  2. Wilson C, 2017, Res Rev J Eng Technol, 6
  3. Kanuri N, 2022, Cureus, 14, e21777
  4. Castro-Manrreza M, 2019, Stem Cells Int, 2019, 4541797
  5. Iwata H, 2022, Int J Pharm X, 4, 100135
  6. Ding H, 2022, Biomater Investig Dent, 9, 75
  7. Emde B, 2014, Eur J Histochem, 58

Cited by

Sorted by citations

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Metadata

Authors
Singh, S., Muniz De Oliveira, F., Wang, C., Kumar, M., Xuan, Y., DeMazumder, D., Sen, C. K., & Roy, S.
Affiliations
  • University of Pittsburgh (US)
  • Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Corresponding author
Roy, Sashwati
Venue
Advances in Wound Care
Published
2025-08-01
Volume
14
Issue
8
Pages
393-408
ISSN
  • 2162-1918 (ISSN-L)
  • 2162-1934
OpenAlex
W4410313918
Cited by
6
References
75
Citations by year
2026: 5 · 2025: 1
FWCI
4.03
Type
journal article
Language
en
Open access
Locations
  • Advances in Wound Care (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Heart, Lung, and Blood Institute (DP2HL157941)
  • National Institute of General Medical Sciences (K25GM143572)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125835)
Topics
  • Health Sciences › Medicine › Surgery › Wound Healing and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords
chronic wounds, biofilm quantification, image segmentation, machine learning, scanning electron microscopy, PNA-FISH, interobserver variability, open source software
MeSH terms
Machine Learning, Chronic Disease, Humans, Microscopy, Electron, Scanning, Software, Wound Infection, In Situ Hybridization, Fluorescence, Biofilms

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