Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients.

Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii (T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529 bp (RE) sequence of T. gondii. The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×105 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment.

Unlike Butylcycloheptylprodigiosin, Isolated Undecylprodigiosin from Streptomyces parvulus Is Not a MDR1 and BCRP Substrate in Multidrug-Resistant Cancers.

The search for new chemotherapeutics unaffected by efflux pumps would significantly increase life expectancy in patients with malignant cancers. In this study, butylcycloheptylprodigiosin and undecylprodigiosin were HPLC-purified and verified, using nuclear magnetic resonance spectroscopy. Cell cytotoxicity and transportation kinetics on multiple-drug resistance (MDR) cells were evaluated. Daunorubicin and butylcycloheptylprodigiosin were less toxic in the MDR1 overexpressing line, but undecylprodigiosin revealed potent toxicity toward MDR1 and BCRP expressing malignant cells. There was no noticeable change in MDR1 and BCRP transcripts during 3 days of treatment with prodiginines. While daunorubicin and mitoxantrone uptake from the cell environment significantly decreased with increasing multidrug resistance up to 46% and 62%, respectively, the accumulation of undecylprodigiosin and to a lesser extent butylcycloheptylprodigiosin in the resistance cells occurred cell- and dose-dependently via a passive diffusion process and were almost equally sensitive to the parent lines. The efflux of xenobiotics commenced immediately with different kinetics in various cells. A greater amount of daunorubicin and mitoxantrone were rapidly thrown out of their corresponding MDR cells in the absence of the specific inhibitor (3.01 and 1.81 dF/min, respectively) and represented functional efflux pumps. MDR pumps did not apparently influence undecylprodigiosin efflux patterns; but butylcycloheptylprodigiosin was partially removed from EPG85.257RDB cells at the rate of 2.66 and 1.41 dF/min in the absence and presence of verapamil, respectively.