Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients.

Michael Frimpong,Emmanuel Akowuah,Richard Odame Phillips,Samuel Asamoah Sakyi,Bernadette Agbavor,Hubert Senanu Ahor

doi:10.3390/diagnostics9040204

Abstract

Isothermal amplification techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for diagnosing Buruli ulcer, a necrotic skin disease caused by Mycobacterium ulcerans, have renewed hope for the molecular diagnosis of clinically suspected Buruli ulcer cases in endemic districts. If these techniques are applied at district-level hospitals or clinics, they will help facilitate early case detection with prompt treatment, thereby reducing disability and associated costs of disease management. The accuracy as well as the application of these molecular techniques at point of need is dependent on simple and fast DNA extraction. We have modified and tested a rapid extraction protocol for use with an already developed recombinase polymerase amplification assay. The entire procedure from “sample in, extraction and DNA amplification” was conducted in a mobile suitcase laboratory within 40 min. The DNA extraction procedure was performed within 15 min, with only two manipulation/pipetting steps needed. The diagnostic sensitivity and specificity of this extraction protocol together with M. ulcerans RPA in comparison with standard DNA extraction with real-time PCR was 87% (n = 26) and 100% (n = 13), respectively. We have established a simple, fast and efficient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.

Highlights

Mycobacterium ulcerans (M. ulcerans) is the causative agent of Buruli ulcer (BU), a deforming skin disease mostly reported in rural communities in most endemic countries
Diagnosis is critical in BU case management, as antibiotic treatment is very effective with a combination of rifampicin and clarithromycin/streptomycin [1,2]
Fifty-eight clinically suspected BU patients were recruited in this study

Summary

Introduction

Mycobacterium ulcerans (M. ulcerans) is the causative agent of Buruli ulcer (BU), a deforming skin disease mostly reported in rural communities in most endemic countries. The Gold standard diagnostic technique for BU is polymerase chain reaction (PCR) targeting the IS2404 insertion sequence of M. ulcerans The deployment of this technique in endemic communities is hindered due to the sophistication required in setting up and logistical constraints of endemic communities. Recombinase polymerase amplification (RPA) [3] and loop-mediated isothermal amplification (LAMP) [5,6,7] have been developed as diagnostic tools for BU. These techniques were proposed as field-friendly diagnostic techniques during a diagnostic conference organized by the Foundation for Innovative New Diagnostics (FIND) and World Health Organization/Neglected Tropical Diseases

Methods

Results

Discussion

Conclusion