Comparative evaluation of beetroot and Mangosteen Peel extracts as natural alternatives to malachite green in the Kato–Katz method

BACKGROUND: The Kato-Katz technique is a widely recommended reference method for diagnosing soil-transmitted helminth (STH) infections. However, the routine use of Malachite green raises concerns regarding toxicity and environmental impact. Plant-derived natural dyes present a potentially safer alternative, nonetheless, objective and reproducible assessments remain limited. AIMS: The objective of this study was to assess the viability of beetroot and mangosteen peel extracts as alternative staining agents to Malachite green in the Kato-Katz method by a combination of expert evaluation and quantitative image analysis. METHODS: Faecal samples positive for STH eggs were analysed by the Kato-Katz technique, stained with 3% Malachite green, 80% beetroot extract, and 80% mangosteen peel extract. The staining quality was evaluated by medical laboratory professionals, assessment utilizing a three-point ordinal scale. The inter-observer agreement was assessed utilizing the intraclass correlation coefficient (ICC). Non-parametric statistical tests for paired samples were utilized to compare staining techniques. Objective picture analysis was conducted utilizing HSV colour characteristics, contrast-to-noise ratio (CNR), and edge strength measurements. RESULTS: Expert visual evaluation demonstrated consistently elevated median scores across all staining techniques, accompanied by substantial inter-observer concordance (ICC = 0.72-0.80). No statistically significant changes in staining quality were detected between natural dyes and Malachite green (p > 0.05). Quantitative picture analysis revealed consistent HSV colour distributions, sufficient object-background contrast, and similar boundary delineation of helminth eggs across staining techniques. CONCLUSION: The extracts of beetroot and mangosteen peel exhibit staining efficacy comparable to that of Malachite green in the Kato-Katz method for detecting helminth eggs. These natural dyes serve as safe, eco-friendly, and diagnostically dependable alternatives that could enhance biosafety in parasitological laboratories, especially in resource-constrained environments.