Light Forge: A Microfluidic DNA Melting-based Tuberculosis Test

Author:

Mbano Ian M1,Mandizvo Tawanda1,Rogich Jerome2,Kunota Tafara T R1,Mackenzie Jared S1,Pillay Manormoney3,Balagaddé Frederick K1

Affiliation:

1. Africa Health Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu Natal, Durban, South Africa

2. University of Massachusetts Medical School, Worcester, MA

3. Medical Microbiology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa

Abstract

Abstract Background There is a well-documented lack of rapid, low-cost tuberculosis (TB) drug resistance diagnostics in low-income settings across the globe. It is these areas that are plagued with a disproportionately high disease burden and in greatest need of these diagnostics. Methods In this study, we compared the performance of Light Forge, a microfluidic high-resolution melting analysis (HRMA) prototype for rapid low-cost detection of TB drug resistance with a commercial HRMA device, a predictive “nearest-neighbor” thermodynamic model, DNA sequencing, and phenotypic drug susceptibility testing (DST). The initial development and assessment of the Light Forge assay was performed with 7 phenotypically drug resistant strains of Mycobacterium tuberculosis (M.tb) that had their rpoB gene subsequently sequenced to confirm resistance to Rifampin. These isolates of M.tb were then compared against a drug-susceptible standard, H37Rv. Seven strains of M.tb were isolated from clinical specimens and individually analyzed to characterize the unique melting profile of each strain. Results Light Forge was able to detect drug-resistance linked mutations with 100% concordance to the sequencing, phenotypic DST and the “nearest neighbor” thermodynamic model. Researchers were then blinded to the resistance profile of the seven M.tb strains. In this experiment, Light Forge correctly classified 7 out of 9 strains as either drug resistant or drug susceptible. Conclusions Light Forge represents a promising prototype for a fast, low-cost diagnostic alternative for detection of drug resistant strains of TB in resource constrained settings.

Funder

The Howard Hughes Medical Institute

University of KwaZulu-Natal College of Health Sciences

Wellcome Trust

Publisher

Oxford University Press (OUP)

Subject

General Medicine

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