Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance: A Starworms study protocol

Johnny Vlaminck, Piet Cools, Marco Albonico, Shaali Ame, Mio Ayana, Jeffrey Bethony, Giuseppe Cringoli, Daniel Dana, Jennifer Keiser, Maria P Maurelli, Antonio Montresor, Zeleke Mekonnen, Greg Mirams, Rodrigo Corrêa-Oliveira, Roger Prichard, Nour Rashwan, Laura Rinaldi, Somphou Sayasone, Eurion Thomas, Jaco J Verweij, Jozef Vercruysse, Bruno Levecke, Johnny Vlaminck, Piet Cools, Marco Albonico, Shaali Ame, Mio Ayana, Jeffrey Bethony, Giuseppe Cringoli, Daniel Dana, Jennifer Keiser, Maria P Maurelli, Antonio Montresor, Zeleke Mekonnen, Greg Mirams, Rodrigo Corrêa-Oliveira, Roger Prichard, Nour Rashwan, Laura Rinaldi, Somphou Sayasone, Eurion Thomas, Jaco J Verweij, Jozef Vercruysse, Bruno Levecke

Abstract

Background: To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs.

Methods: A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the β-tubulin gene. These SNPs are known to be linked to AR in animal STHs.

Discussion: The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the β-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs.

Trial registration: The trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488).

Conflict of interest statement

The Mini-FLOTAC apparatus has been developed and is patented by GC, but the patent will be handed over to the University of Naples Federico II. The FECPAKG2 technology was developed and patented by Techion Group Ltd, of which GM and ET are employees. These affiliations however did not play any role in the preparation and submission of this study protocol. All other authors declare that they have no competing interests.

Figures

Fig 1. A schematic overview of the…
Fig 1. A schematic overview of the different steps of the field trials.

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