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 104 and 49% versus 23% at a concentration of 102. 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.

Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats.

PURPOSE: The relationship between the genetic diversity of Blastocystis and immune surveillance in precancerous colons with blastocystosis is still under investigation. This study aimed to identify the genetic Blastocystis variants among 54 symptomatic human isolates and their relationship to mucosal immune surveillance in the precancerous polyps of experimentally infected rats. METHODS: Polymerase chain reaction and high-resolution melting (PCR/HRM) curves discriminated human symptomatic Blastocystis isolates into subtypes (STs)/intrasubtypes, which were orally administered to rats to induce experimental infection. Then, the mucosal immune responses of the infected colons were evaluated in relation to polyp formation through immunostaining to identify mucus MUC2 and determine mucosal immune cell (goblet, lymphocyte and mast) counts, secretory IgA levels and parasitic intestinal invasion. RESULTS: ST1, ST3, and ST4 were found in 18.5% (10/54), 54.7% (29/54), and 27.8% (15/54) of the samples, respectively. Then, the HRM curve discriminated ST3 into the wild, mutant, and heterozygous [17/54 (31.5%), 5/54 (9.3%), and 7/54 (12.9%)] intrasubtypes. ST1 and ST4 had no genetic variations. Precancerous polyps were detected in the colons of 40.5% of the infected rats. ST1 constituted 14.7% of these cases, while the wild, mutant, and heterozygous intrasubtypes of ST3 showed polyps in 12.9%, 5.5%, and 5.5% of cases, respectively. Only 1.9% of the polyps were related to ST4. MUC2 showed weak immunostaining in 44.5% of the infected colons, and 38.9% were polyp inducers. Low goblet cell numbers and high interepithelial lymphocyte counts were significantly associated with polyp formation, particularly with ST1 and wild ST3. Among the polyp inducers, high numbers of mast cells were detected in wild ST3 and ST4, while a low number was found with heterozygous ST3. The level of secretory IgA was low in polyp-inducing STs. Most of the results were statistically significant. CONCLUSION: Immunosurveillance showed a potential relationship between ST1 and the ST3 intrasubtypes and precancerous polyps. This relationship may provide insight into the prevention and/or development of new immunotherapeutic strategies to combat colorectal cancer.

Antiprotozoal activity of magnesium oxide (MgO) nanoparticles against Cyclospora cayetanensis oocysts.

Outbreaks of Cyclospora cayetanensis infection have been linked to consumption of food and water contaminated by oocysts that can survive both physical and chemical disinfectants. Magnesium oxide (MgO) nanoparticles (NPs) can be potentially used in food as bactericides. In this study, C. cayetanensis pre- and post-sporulated oocysts were exposed to MgO NPs with different doses ranging from 1.25-25 mg/ml. With comparison to control, the antiprotozoal activity of MgO NPs was evaluated by identifying the median effective concentration dose (EC50), lethal concentration dose (LC90), microscopically changes on treated oocysts and rates of sporulation. Among pre- and post-sporulated oocysts, MgO NPs ≥ EC50 was observed after 24 h at concentrations 10 and 12.5 mg/ml, respectively, while ≥ LC90 was observed after 24 h, 48 h and 72 h at concentrations 15, 12.5 and 10 mg/ml, respectively. MgO NPs treated oocysts showed abnormal morphological changes such as an increase in size, wall injury, deposition of vacuolated homogenous particles in the cytoplasm, evacuation of oocyst's contents, and collapse. Sporocysts of treated oocysts were noticed to be peripherally shifted. Sporulation failure of treated oocysts achieved ≥90% after 24 h and 72 h of incubation with 15 and 12.5 mg/ml, respectively, while it was 10.1% among non-treated. All the differences were statistically significant. Our results demonstrated that MgO NPs has a significant anti-Cyclospora effect on both unsporulated and sporulated oocysts, especially considering that it could be biologically synthesized, that way it can be used safely as a preventive agent in food and water disinfectant treatment.