Data_Sheet_1_Highly sensitive quantitative phase microscopy and deep learning aided with whole genome sequencing for rapid detection of infection and antimicrobial resistance.docx

Current state-of-the-art infection and antimicrobial resistance (AMR) diagnostics are based on culture-based methods with a detection time of 48–96 h. Therefore, it is essential to develop novel methods that can do real-time diagnoses. Here, we demonstrate that the complimentary use of label-free optical assay with whole-genome sequencing (WGS) can enable rapid diagnosis of infection and AMR. Our assay is based on microscopy methods exploiting label-free, highly sensitive quantitative phase microscopy (QPM) followed by deep convolutional neural networks-based classification. The workflow was benchmarked on 21 clinical isolates from four WHO priority pathogens that were antibiotic susceptibility tested, and their AMR profile was determined by WGS. The proposed optical assay was in good agreement with the WGS characterization. Accurate classification based on the gram staining (100% recall for gram-negative and 83.4% for gram-positive), species (98.6%), and resistant/susceptible type (96.4%), as well as at the individual strain level (100% sensitivity in predicting 19 out of the 21 strains, with an overall accuracy of 95.45%). The results from this initial proof-of-concept study demonstrate the potential of the QPM assay as a rapid and first-stage tool for species, strain-level classification, and the presence or absence of AMR, which WGS can follow up for confirmation. Overall, a combined workflow with QPM and WGS complemented with deep learning data analyses could, in the future, be transformative for detecting and identifying pathogens and characterization of the AMR profile and antibiotic susceptibility.</p

Nucleotide polymorphisms in the sequences of putative virulence factors found in the genome of <i>Clavibacter michiganensis</i> subsp. <i>nebraskensis</i> strains GIL1 and HF4. PCR primers flanking each locus were used in sequencing reactions to obtain the complete sequence of each gene.

<p>Sequences were aligned and examined for nucleotide sequence differences. Primers were designed from reference strain NCPPB 2581 genomic sequence.</p><p>Nucleotide polymorphisms in the sequences of putative virulence factors found in the genome of <i>Clavibacter michiganensis</i> subsp. <i>nebraskensis</i> strains GIL1 and HF4. PCR primers flanking each locus were used in sequencing reactions to obtain the complete sequence of each gene.</p

Mean aggressiveness (n = 12 plants) of 28 <i>Clavibacter michiganensis</i> subsp. <i>nebraskensis</i> strains estimated from foliar blight severity on maize hybrid DKC 55–09.

<p>Plants were inoculated at the V3–V4 crop developmental stage and foliar blight severity was rated as the proportion of leaf area affected six days after inoculation. Bars represent standard errors of the mean. *Strain 91-R was included as a reference for pathogenicity in this experiment.</p

Examples of colony morphology after 5 days growth on nutrient broth yeast extract (NBY) medium of 37 putative isolates of <i>Clavibacter michiganensis</i> subsp. <i>nebraskensis</i>.

<p>A small mucoid orange colony; B large mucoid orange colony; and C large fluidal yellow colony.</p

Supplementary document for Towards Ultrafast Quantitative Phase Imaging via Differentiable Microscopy - 6605102.pdf

Supplemental Documen

Supplementary document for Towards Ultrafast Quantitative Phase Imaging via Differentiable Microscopy - 6785254.pdf

Supplemental Documen

TIRF1.avi

Excitation of multiple modes inside the optical waveguide and resulting TIRF image obtained from superposition of 250 images