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

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. 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. 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. 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. 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.