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 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 °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.

A Systematic Review on Suitability of Molecular Techniques for Diagnosis and Research into Infectious Diseases of Concern in Resource-Limited Settings

“…A TPHD-RPA assay, which is based on the RPA technology, was developed by Frimpong et al [ 56 ] to simultaneously and rapidly detect H. ducreyi and T. pallidum . The genes targeted were pol A for yaws and the hemolytic cytotoxin HhdA gene for H. ducreyi.…”

“…The genes targeted were pol A for yaws and the hemolytic cytotoxin HhdA gene for H. ducreyi. The assay was demonstrated to have 94–95% and 100% sensitivity and specificity, respectively [ 56 ] (Frimpong et al, 2020). RPA technology appears to have some advantage over LAMP in that it has a shorter turn-around time (15 min as compared to 30 to 60 min in the case of LAMP) at 37 to 42 degrees [ 57 , 58 ].…”

“…Typical turn-around time for LAMP assays is short as compared to conventional PCR. Specifically, it takes about 30 min to one hour at 60 to 65 °C [ 56 , 161 ]. Furthermore, the method uses more robust reagents that can easily be transported under suboptimal conditions [ 161 ].…”

“…Furthermore, the method uses more robust reagents that can easily be transported under suboptimal conditions [ 161 ]. However, one disadvantage associated with LAMP assays generation is that it requires the design of four to six primes per assay, which can be challenging, even though that increases the specificity of the assay [ 56 , 162 ]. Invariably, these examples demonstrate that the development of effective isothermal (NAAT) assays is a step in the right direction due to their relative ease of use in low-resource settings for differential disease diagnoses, monitoring drug resistance, as well as general disease screening.…”

LAMP4yaws:Treponema pallidum,Haemophilus ducreyiloop mediated isothermal amplification − protocol for a cross-sectional, observational, diagnostic accuracy study

“…A recombinase polymerase amplification test for TP and HD has also recently been developed. 24 Similarly, this test relies on isothermal amplification, which can be conducted close to the patient and has performed well in a laboratory evaluation. Pursuing a clinical evaluation in a programmatic setting will also be useful.…”