# iFIND INH/FQ: a LC-aNAAT assay for rapid simultaneous detection of isoniazid and fluoroquinolone resistance in Mycobacterium tuberculosis

## TL;DR

The iFIND INH/FQ test is a fast and accurate molecular tool for detecting drug resistance in tuberculosis, suitable for use in low-resource labs.

## Contribution

The iFIND INH/FQ is a novel low-complexity assay for rapid, simultaneous detection of isoniazid and fluoroquinolone resistance in tuberculosis.

## Key findings

- The assay detected INH resistance with 97.59% sensitivity and 98.10% specificity.
- For FQ resistance, the assay showed 92.16% sensitivity and 97.33% specificity for levofloxacin.
- The test achieved a limit of detection of 20.79 CFU/mL for INH and 9.34 CFU/mL for FQs.

## Abstract

To evaluate the performance of the iFIND INH/FQ, a low-complexity molecular assay, for the rapid and simultaneous detection of resistance to isoniazid (INH) and fluoroquinolones (FQs) in Mycobacterium tuberculosis. Frozen sputum specimens stored at the Chengde Center for Disease Control and Prevention laboratory were used. Phenotypic drug susceptibility testing (pDST) and DNA sequencing served as reference standards. The limit of detection (LOD) was determined using probit regression with spiked samples. The assay’s ability to detect resistance-conferring mutations in katG, inhA, and gyrA genes was assessed using genotypically characterized strains. Diagnostic accuracy was evaluated against pDST. The LOD was 20.79 CFU/mL for INH and 9.34 CFU/mL for FQs. The assay detected all targeted mutations except ahpC c.-6 associated with INH resistance. Compared to pDST, the iFIND INH/FQ assay demonstrated a sensitivity of 97.59% (95% confidence interval [CI]: 91.63–99.34%) and specificity of 98.10% (95% CI: 94.57–99.35%) for INH resistance. For FQ resistance, sensitivity was 92.16% (95% CI: 81.50–96.91%) for levofloxacin and 92.00% (95% CI: 81.16–96.85%) for moxifloxacin, with specificities of 97.33% (95% CI: 93.89–98.85%) and 96.81% (95% CI: 93.21–98.53%), respectively. Sequencing confirmed iFIND results in the majority of discrepant cases (100% for INH and 55.65% for FQs). The iFIND INH/FQ LC-aNAAT is a highly accurate and rapid molecular assay for simultaneous detection of INH and FQ resistance. It is a promising tool for scaling up rapid drug susceptibility testing in clinical and peripheral laboratory settings.

As a low-complexity automated nucleic acid amplification test, the iFIND assay achieves the goal of simultaneously detecting isoniazid and fluoroquinolone resistance in approximately 90 min, perfectly meeting the TPP's core requirements for “rapid” and “simple operation.” Its fully integrated system minimizes manual steps and contamination risk, making it highly suitable for use in resource-limited, lower-biosafety-level primary laboratories.