Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

Abstract

Yaws is a skin debilitating disease caused by <i>Treponema pallidum</i> subspecies <i>pertenue</i> with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as <i>Haemophilus ducreyi</i> (<i>H. ducreyi</i>). We have developed a RPA assay for the simultaneous detection of <i>Treponema pallidum</i> (<i>T. pallidum</i>) and <i>H. ducreyi</i> (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of <i>T. pallidum</i> and <i>H. ducreyi</i> within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for <i>T. pallidum</i> and <i>H. ducreyi</i>, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both <i>T. pallidum</i> and <i>H. ducreyi</i> in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.