Biochemical and Parasitological Studies on the Effect of hUCB-Selected CD34+ Progenitor/Stem Cells in Mice Infected with Schistosoma mansoni

BACKGROUND AND OBJECTIVES: Placenta and blood that remained in the umbilical cord is routinely available as a discarded tissue after deliveries and it is free of any legal, moral, ethical or religious objections, providing a high number of multipotent CD34+ progenitor and stem cells. Using ex vivo isolated CD34+ cells from human umbilical cord blood (hUCB) have emerged as promising candidates to treat various diseases, including exogenous pathogenic infections. We have expanded to build a rational approach to study the effect of CD34+ cells after damaged liver tissues by the devastating human parasitic flatworm Schistosoma mansoni. METHODS AND RESULTS: Experimental studies were conducted in the Department of Zoology, Faculty of Science and Departments of Parasitology and Physiology, Faculty of Medicine, SCU, Egypt. We have studied the impact of ex vivo preparation of CD34+ cells from hUCB on S. mansoni-induced liver fibrosis de novo, and treated for shorter and longer periods in vivo. Ova count, ALT and albumin were measured at specific time interval and histopathological examination of liver was conducted to confirm the biochemical results. The data obtained were statistically analyzed by ANOVA between groups. It was found that the administration of CD34+ cells have modestly reduced liver damage; reduced the S. mansoni infection associated elevation in serum levels of ALT; significantly improved serum levels of albumin and reduced egg granuloma diameter in the livers. CONCLUSIONS: We demonstrated that CD34+ cells can markedly ameliorated liver fibrosis in vivo and may be beneficial for therapy to recover organ structure and/or function of S. mansoni-infected mice.

In vitro and in vivo effects of Quillaja saponaria on Blastocystis hominis isolates

Blastocystis hominis is a protozoan parasite that inhabits the human intestinal tract. Various epidemiological surveys have recorded 50-60% prevalence in developing countries. Until now, the most commonly recommended drug is metronidazole [MTZ] which can cause undesirable side effects and failures in treatment. To investigate the in vitro and in viva effects of Quillaja saponaria [QS] against clonal cultures of B. hominis and to demonstrate its implemented ultrastructural changes. Two fresh stool isolates of B. hominis were processed for in vitro cultivation using Jones media. In comparison to MTZ, different concentrations of QS were added to assess its lethal dose; QS [500 micro g] was used to assess programmed cell death for both isolates, using transmission electron microscopy [TEM]. Experimental infection of rats was performed to assess QS induced intestinal histopathological changes as compared to treatment with MTZ. With isolate I. QS [1000 micro g/ml] produced a high significant reduction [P

Evaluation of DNA melting curve analysis real-time PCR for detection and differentiation of cryptosporidium species

Cryptosporidium is a leading cause of persistent diarrhea in developing countries where it is reported to be more common in malnourished children with more severe consequences of the disease than in well nourished ones. PCR which is the most sensitive and specific diagnostic method for detecting and genotyping Cryptosporidium still has some drawbacks. The present study aimed at evaluating melting curve analysis after real-time PCR in diagnosis and genotyping Cryptosporidium as an alternative to traditional multiplex PCR followed by gel electrophoresis. Seventeen naturally infected immun°Compromised patients suffering from Cryptosporidium diagnosed by both microscopy and immun°Chrommatographic strip assay provided the study isolates. All samples were subjected to traditional multiplex PCR followed by gel electrophoresis and real-time PCR followed by melting curve analysis. Melting curve analysis real-time PCR proved 100% sensitivity and specificity when compared to traditional multiplex PCR. It had the advantage of being less time consuming [1 hr], less liable for post amplification contamination by carry-over as the pr°Cess is completed in a closed tube. Genotyping of Cryptosporidium on the basis of melting curve analysis revealed that C. hominis showed two distinct peaks at 85.5°C and 88.5°C while C. parvum showed two distinct peaks at 80.1°C and 88.5°C. Interestingly, one isolate proved to be a mixed infection showing three peaks at 80.1°C, 85.5°C and 88.5°C. DNA melting curve analysis real-time PCR offers a step forward in the detection and differentiation of Cryptosporidium spp. by circumventing disadvantage of traditional PCR

Comparison of polymerase chain reaction, immunochromatographic assay and staining techniques in diagnosis of cryptosporiodiosis

Cryptosporidiosis represents a major health problem worldwide. In developed countries, massive outbreaks have been reported while in developing countries, it is associated with significant morbidity and mortality, especially among infants and children. Although the modified acid-fast technique is the commonly used slain for its detection, its sensitivity and specificity appeared to be rather low. The present study aimed at comparing the conventional diagnostic method with the recent techniques namely immunochromatographic [ICT] strip assay and multiplex allele specific polymerase chain reaction [MAS-PCR]. The second objective was to genotype the diagnosed isolates using MASPCR. Seventy six immunocompromised patients having acute or chronic diarrhea were selected from the attendance of the pediatrics, oncology and nephrology clinics in Suez Canal University Hospital. Cryptosporidiosis was diagnosed by Kinyoun acid fast stain, ICT strip assay and MAS-PCR. Samples proved positive for cryptosporidiosis were genotyped using MAS-PCR. Using MAS-PCR as Gold standard method, modified Kinyoun acid fast stain and ICT strip showed sensitivity [79 vs 89%], specificity [98 vs 100%], positive predictive value [94 vs 100%], negative predictive value [93 vs 100%] and diagnostic accuracy [88.5 vs 94.5%]. Using MAS-PCR for genotyping, C. parvum comprised the majority [68.4%] of cases while C. hominis was only 26.3%. Only one patient had mixed genotype infection. C. parvum infections were associated with low intensity of oocyst shedding while C hominis infections were with high intensity of oocyst shedding. The agreement between microscopy and MAS-PCR results proved that only 60% of positive cases identified as C. hominis [type 1] by MASPCR were positive by microscopy while, 92.3% of C. parvum [type II] positive cases by MAS-PCR were positive by microscopy. The agreement between ICT strip and MAS-PCR results proved that the strip identified 100% of positive cases of C. hominis [type I] and 84.6% of C. parvum [type II] positive cases by MAS-PCR. The ICT strip assay gave very good results regarding performance and came second to MASPCR in ranking which has an additional advantage due to its ability to genotype diagnosed isolates. The low sensitivity of staining method and high cost of MAS-PCR recommend the ICT strips for the wide use especially in field of diagnosis and in outbreaks where large number of tests needs to be performed in a short period of time