# Comprehensive validation of the rapid tick exposure test (RaTexT®): accuracy, reproducibility and comparative performance to detect acaricide resistance in livestock ticks

## TL;DR

RaTexT® is a fast, on-site test for detecting acaricide resistance in ticks, shown to be accurate and reliable with minimal replication.

## Contribution

The study introduces a novel comparison of RaTexT® with the adult immersion test (AIT), overcoming previous limitations.

## Key findings

- A single RaTexT® strip achieved 91.3% agreement with box classification, showing high consistency.
- Monte Carlo simulations showed diagnostic accuracy increased from 81.2% to 86.1% with more ticks per compartment.
- RaTexT® matched AIT in detecting deltamethrin resistance but showed lower mortality for cypermethrin/chlorpyriphos/PBO.

## Abstract

RaTexT®, an innovative rapid tick exposure test, was recently developed to provide farmers with quick, on-site results to improve their acaricide resistance management. This was achieved by exposing partially engorged adult ticks to a specially designed acaricide-impregnated matrix fitted inside a transparent polypropylene box. Each RaTexT® box contains six strips of four small, interconnected compartments, in which ticks are exposed immediately after removal from cattle in the field. In this study, we assessed whether a single strip of four interconnected compartments, instead of six strips, was sufficient to accurately detect resistance to deltamethrin and to a combination of cypermethrin, chlorpyrifos and piperonyl butoxide (PBO). We also statistically analysed the optimal number of ticks per compartment (ranging between 5 and 8). Moreover, the test reproducibility was checked by two independent observers who counted dead and live ticks in each compartment. Finally, a comparative analysis was undertaken between adult ticks exposed in RaTexT® and in the adult immersion test (AIT), and also with larvae in the resistance intensity test (RIT) and in the larval packet test (LPT). The novelty of this study lies in comparing adult ticks exposed in RaTexT and in the AIT Test, thereby overcoming limitations of previous studies, in which adults in RaTexT were compared with larvae in the LPT.

The internal coefficient of variation (CV) was calculated for each dose, box and acaricidal product to assess within-box consistency. The effect of the number of ticks per compartment (n = 5–8) was examined using Monte Carlo simulations. Inter-observer reliability of reading RaTexT® was statistically measured using Cohen’s kappa coefficient (κ). The comparative performance analysis of the bioassays was conducted using generalised linear models (GLMs) with laboratory and field strains of Rhipicephalus microplus ticks in Brazil.

Overall agreement between individual strips and their corresponding box classification was 91.3%, indicating high consistency between replicates. The predefined threshold of  ≥  90% accuracy was met by a single strip, supporting the test's robustness even with minimal replication. The internal coefficient of variation within each RaTexT® box was high for deltamethrin (1.285 at 1× dose, 1.109 at 5× dose and 1.268 at 10× dose), but lower for cypermethrin/chlorpyriphos/PBO (0.648 at 1, 0.305 at 5× dose and 0.194 at 10× dose). Variability in the controls was relatively high (CV 1.776). Monte Carlo simulations showed that diagnostic accuracy gradually increased from 81.2% with five ticks to 86.1% with eight ticks per compartment. Furthermore, there was substantial agreement between the mortality of ticks assessed by two independent observers (κ = 0.664). Finally, the comparative test analysis revealed that the deltamethrin resistance level in RaTexT® matched that observed in the AIT. Resistance to deltamethrin was also confirmed by the LPT, with resistance ratios (RR) of 33.8 and 39.5 for two different field strains (Biotech and UFRRJ, respectively). For cypermethrin/chlorpyriphos/PBO, RaTexT® exhibited significantly lower mortality than the AIT. Resistance was also confirmed by LPT, with RR of 5.2 for Biotech strain and 7.2 for the UFRRJ strain.

Overall, these findings demonstrate that RaTexT® is accurate and reproducible with a single test strip, making it a practical and cost-effective test that complements traditional laboratory bioassays.