Rapid detection of Toxoplasma gondii DNA in cat feces using colorimetric loop-mediated isothermal amplification (LAMP) assays targeting RE and B1 genes.

Toxoplasma gondii is an obligate protozoan parasite that can infect mammals and birds. Cats are the definitive host of T. gondii and have a very important role in transmission of toxoplasmosis due to the shedding of millions of unsporulated oocysts, that become infective in the environment. Since cats play a major key role in the epidemiology of toxoplasmosis, rapid and accurate diagnosis of infected cats has utmost importance. In this study, we developed a novel colorimetric loop mediated isothermal amplification (LAMP) assay detecting T. gondii RE gene and modified a previously developed colorimetric LAMP assay targeting B1 gene to detect T. gondii DNA in cat feces for the first time. The analytical sensitivity of colorimetric LAMP assays was determined using plasmid controls. The clinical sensitivities of both colorimetric LAMPs were determined using cat fecal DNA samples that were confirmed to be positive by two different real-time PCRs in our previous study. According to the results, analytical sensitivities of both assays were 1 copy plasmid/reaction. Using real-time PCR as a reference method, sensitivities of colorimetric LAMP assays targeting RE and B1 genes were 100% and 97.56% whereas specificities of both assays were 100%. Overall, the colorimetric LAMP RE assay developed in this study brings an advantage in the diagnosis of T. gondii in cat fecal samples since it has higher sensitivity, does not need for experienced personnel, and can be applied in basic laboratories or in the field.

Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor.

BACKGROUND: Cryptosporidium spp. is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp. can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp. in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp. prevalence was determined using Ziehl-Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp. in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl-Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl-Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

GRA8 DNA vaccine formulations protect against chronic toxoplasmosis.

Toxoplasma gondii has a very wide host range and infects all warm-blooded animals including humans. The disease causes great economic losses both in animals and humans. Vaccination is the most effective approach to fight against toxoplasmosis however an effective vaccine has not been developed yet. In the present study, GRA8 protein of T. gondii that showed high immunogenicity in our previous microarray screening study was used to develop a DNA vaccine using pcDNA 3.3 vector for the first time. In order to increase the potency of the DNA vaccine, 10 times lower amount of GRA8 DNA vaccine was combined with molecular adjuvant CpG and formulated into a commercial liposome (pcDNA3.3-GRA8+CpG+Escort). Mice were vaccinated intramuscularly two times at three-week intervals and challenged orally with the T. gondii PRU strain tissue cysts. The humoral immune response was determined by Western Blot and ELISA. The cellular immune response was analyzed by flow cytometry, cytokine ELISA and MTT assay. Among the vaccine groups, pcDNA3.3-GRA8 and pcDNA3.3-GRA8+CpG+Escort induced strong IgG response compared to controls (P < 0.001). The IgG1 and IgG2a responses showed a balanced Th1-Th2 polarization. The ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.01). The amount of tissue cysts in the group of mice vaccinated with pcDNA3.3-GRA8 decreased significantly compared to control groups (P < 0.0001). In the group vaccinated with pcDNA3.3-GRA8+CpG+Escort, the amount of tissue cysts also decreased significantly compared to PBS (P = 0.0086) and Empty plasmid+CpG+Escort (P = 0.0007) groups. This study showed for the first time that pcDNA 3.3. vector encoding GRA8 with or without CpG and Liposome can induce strong cellular and humoral immune responses and confer strong protection against mouse model of chronic toxoplasmosis.