Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl–Neelsen Stain

(1) Background: The wet mount staining technique is a simple, economical, and rapid method for detecting parasitic stages. The objective of the current study was to evaluate wet mount diagnostic stains for Cryptosporidium infection in human faecal samples and to compare the sensitivity and qualitative performance of toluidine blue (TolB) and modified Ziehl–Neelsen (mZN) stain. (2) Methods: The collection, purification, and molecular amplification of Cryptosporidium oocysts were performed. TolB, malachite green, trypan blue, and crystal violet were evaluated qualitatively to diagnose Cryptosporidium oocysts. The outperforming stain was compared to mZN using a quantitative evaluation and qualitative scoring system. (3) Results: The oocysts of Cryptosporidium parvum were effectively purified and utilized for spiking. TolB was the most effective diagnostic stain among wet mount stains for detecting Cryptosporidium oocysts. TolB outperformed mZN in terms of sensitivity, with 100% versus 79% at a concentration of 10(4) and 49% versus 23% at a concentration of 10(2). TolB had the most favourable qualitative stain characteristics against mZN; however, sample freshness and being a temporary stain were crucial considerations. (4) Conclusions: This study emphasizes that TolB, as a routine wet mount technique for screening Cryptosporidium infection, will provide a more sensitive and faster method than mZN staining.