Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in yaws-like lesions

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue 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 Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 &deg
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 T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi 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.