# Single-cell based diagnostic platform with single-molecular transcriptional response profiling for rapid phenotypic antimicrobial susceptibility testing of gonorrhea

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2024 · $796,454

## Abstract

PROJECT SUMMARY
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is in the top tier of AMR threats as defined by
WHO. NG is responsible for the second most prevalent sexually transmitted infection worldwide, which can
cause long-term health consequences. Unfortunately, NG has rapidly developed resistance to all first-line
antimicrobials previously recommended for the treatment of gonorrhea, including the last viable antimicrobial,
ceftriaxone. As AMR continues to evolve, there is an urgent need for personalized treatment approaches that
can spare the use of ceftriaxone to reduce the chances of NG becoming resistant to this antimicrobial. However,
this requires clinicians to know drug resistance or susceptibility quickly enough to inform prescription decisions.
The Centers for Disease Control and Prevention (CDC) periodically publishes STD treatment guidelines to help
clinicians, which are informed by susceptibility data generated by the national CDC Gonococcal Isolate
Surveillance Project (GISP). However, determining AMR requires prolonged (24-48 hours) microbiological
cultivation in sophisticated laboratory facilities, which has been supplanted by nucleic acid amplification tests
(NAAT) for diagnosis of NG infections. The widespread adoption of NAAT has created a critical void in AMR
testing, leading to a loss of capability to perform culture of NG in most testing clinics.
We propose to develop a rapid AST platform for NG, capable of testing against multiple antimicrobial conditions
at scale directly from clinical specimens. Our proposed method involves measuring the earliest transcriptional
responses to antimicrobials for rapid AST. We and others have shown that measuring mRNA expression levels
following a 10-minute antimicrobial exposure can predict susceptibility well before phenotypic changes in growth
are observable. This forms the basis of our proposed single-cell mRNA-enabled AST (sc-mRNA-AST) for NG.
To achieve sc-mRNA-AST for NG, our platform will use droplet microfluidics to isolate and analyze individual NG
cells from clinical specimens with heterogeneous bacterial flora. We will first identify the most susceptibility-
informative transcripts agnostic to mechanisms of resistance against each of 3 NG-relevant antimicrobials. We
will use ultrasensitive single-molecule fluorescence spectroscopy to quantify mRNA molecules from individual
cells without the need for amplification. Additionally, we will incorporate an assembly-line-like, cascaded
microfluidic design into our platform, allowing for numerous sc-mRNA-AST assays against clinically relevant
antimicrobial conditions. With an additional 2 minutes per condition, our platform can test 15 antimicrobial
conditions to determine susceptibility and minimal inhibition concentrations (MICs) for 3 antimicrobials within 1
hour, directly from clinical urogenital samples. To evaluate the performance of our platform, we will use contrived
samples with NG isolates and prospectively co...

## Key facts

- **NIH application ID:** 10824953
- **Project number:** 1R01AI181217-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Tza-Huei Jeff Wang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $796,454
- **Award type:** 1
- **Project period:** 2024-06-17 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10824953

## Citation

> US National Institutes of Health, RePORTER application 10824953, Single-cell based diagnostic platform with single-molecular transcriptional response profiling for rapid phenotypic antimicrobial susceptibility testing of gonorrhea (1R01AI181217-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10824953. Licensed CC0.

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