simple and rapid method for the detection of HIV-1/HCV in co-infected patients

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

[TGF-b downregulation by RNAi technique in ex vivo-expanded HSCs on 3D DBM scaffold]

Bone Marrow Transplantations [BMT] are limited by low CD34+ cell counts in umbilical cord blood [UCB] and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells [HSCs] by enhancing their self-renewal activity on demineralized bone matrix [DBM] scaffold coated with mesenchymal progenitor cells [MPCs] and unrestricted somatic stem cells [USSCs] was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed. USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting [MACS] method and were transfected by siRNA against TGFbR2 in two-dimensional [2D] and three-dimensional [3D] cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated. Ex vivo expansion of CD34+ cells was significantly enhanced [41 +/- 0.7 folds] by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay. The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer