# External quality assessment for yaws elimination in low- and middle-income countries using plasmid-based proficiency test items

## TL;DR

This study developed a plasmid-based quality control program to improve yaws testing in low-resource labs, ensuring accurate diagnosis and supporting elimination efforts.

## Contribution

A plasmid-based external quality assessment program was developed for yaws diagnostics in resource-limited settings.

## Key findings

- Plasmid-based PTIs were stable under dry field conditions with no significant copy number loss.
- Reference labs achieved 95.0-100.0% qPCR concordance, while district labs had 76.0-100.0% LAMP concordance.
- Retesting of field samples showed 100% correct identification of TP and HD by African reference labs.

## Abstract

We aimed to establish an external quality assessment (EQA) programme for the yaws eradication campaign that would meet the needs of reference and district-level laboratories in low- and middle-income countries.

We designed proficiency testing items (PTIs) using a plasmid containing gene target sequences for Treponema pallidum (TP) and Haemophilus ducreyi (HD). The storage stability of the plasmids under different environmental conditions was then tested. A proficiency testing panel of seven swabs loaded with different concentrations of plasmids in different combinations, as well as human HEK293 cells to simulate the sample background, was prepared and sent to participating reference (RL) and district (DL) laboratories in Ghana, Côte d’Ivoire and Cameroon followed by three rounds of blinded proficiency testing. We tested quantitative real-time PCR (qPCR) performance of reference laboratories and loop-mediated isothermal amplification (LAMP) performance of district laboratories and retested 20% of human field samples at the London School of Hygiene & Tropical Medicine laboratories to further assess qPCR quality.

PTIs proved to be stable in dry conditions with no significant loss of copy number. Participating laboratories achieved qPCR results with a concordance of 95.0-100.0% (97.7% ± 5.2% (mean±standard deviation ((SD)) with the provider and a concordance of 76.0-100.0% (TP: 90.3 ± 13.7% and HD: 78.5 ± 7.5% (mean±SD)) for LAMP results, with inconsistencies, particularly in the detection of low HD plasmid DNA levels combined with high TP plasmid copies. Retesting of field samples resulted in 100% correct TP and HD sample identification by the African reference laboratories.

We have developed a functional plasmid-based EQA programme specifically designed to meet the needs of resource-poor settings in the tropics. The programme is suitable as a blueprint for other disease programmes.

Yaws caused by the bacterium Treponema pallidum pertenue is a Neglected Tropical Disease and remains a public health challenge in many low- and middle-income countries, where reliable laboratory diagnostics are crucial for successful elimination campaigns. The detection of yaws relays on molecular testing (Nucleic Acid Amplification Tests such as PCR) because serology alone cannot distinguish between yaws and other infections such as syphilis caused by T. pallidum pallidum. Furthermore, the pathogen is considered uncultivable, which excludes traditional microbiological techniques and explains the shortage of whole organism material that could potentially be used as proficiency testing items (PTIs). Our study presents the development and evaluation of a plasmid-based external quality assessment (EQA) programme tailored to reference and district-level laboratories in Ghana, Côte d’Ivoire, and Cameroon. By designing PTIs that mimic clinical samples, we ensured robust assessment of both quantitative real-time PCR (qPCR) in reference laboratories and loop-mediated isothermal amplification (LAMP) in district laboratories. Our findings highlight the stability of PTIs under field conditions and the overall high concordance rates between participant laboratories and the provider, reinforcing the feasibility of implementing such an EQA programme in resource-poor settings.