Role of Fecal Microbiota in Predicting Graft Rejection and Sepsis Among Recipients of Living Donor Liver Transplant in First Year.

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Primary Objective: To characterize the fecal microbiota changes over one year after living donor liver transplantation and its association with graft rejection and sepsis. Secondary objectives : Incidence of graft dysfunction attributable to sepsis, rejection and CMV infection in first year of LDLT....

Primary Objective: To characterize the fecal microbiota changes over one year after living donor liver transplantation and its association with graft rejection and sepsis. Secondary objectives : Incidence of graft dysfunction attributable to sepsis, rejection and CMV infection in first year of LDLT. To study the pre-LT immunological profile and its correlation with post-transplant rejection, immunosuppression requirements and restoration of immunosenescence. To correlate the dynamic changes in immune cells and cytokines in predicting graft rejection and sepsis. STUDY DESIGN Type of study - Single centre, prospective, observational study Study population - consecutive 100 patients fulfilling the eligibility criteria and undergoing living donor liver transplant in ILBS between April 2020 to March 2022. Study duration - 24 months from the date of approval of IEC Methodology: Identification of gut microbiota: DNA extraction All subjects will undergo serum and stool collection the same day. About 5-8g of fecal materialwill be collected .The QIAamp Fast DNA Stool Mini Kit for stool samples will be used for DNA extraction. PCR Amplification of the 16s RNA Gene Briefly, PCR amplification of the V3-V4 hypervariable regions will be done by using the 341F and 806R primers containing the sample specific barcode. Purified amplified product (QIAGEN), will be run on the MiSeq platform with custom sequencing primer reads 1 (R1), read 2 (R2) and index. Whole blood: Whole blood will be (5-10ml) will be collected. Plasma will be separated by centrifugation at 2500rpm. Plasma will be stored at -80C in biobank (NLDB) until processed for cytokines array. Remaining blood will be treated with RBC lysis buffer and will be processed for immune cells analysis. Immunophenotyping for frequency/absolute number of immune cells: The cells in the whole blood will be stained with specific flurochrome labelled monoclonal antibodies for the surface markers for T cells subsets (Th1,Th2, Th17, Tregs), B regulatory cells using Flowcytometry and analysis will be done by FCS express software. Cytokine Array: The pro-inflammatory (IL-1?) and anti-inflammatory cytokines (IL-10)will be measured in plasma of the patients under study. The cytokine array kit will be procured commercially and will be done according to the manufacturer's instructions. STATISTICAL ANALYSIS Data will be reported as mean + SD Categorical variables will be compared using the chi-square test or Fisher exact test Normal continuous variables will be compared using the Student t test Non normal continuous variables will be compared using the Mann-Whitney rank-sum test (unpaired data) or the Wilcoxon test (paired data). Comparison between groups on quantitative variables will be analyzed by ANOVA A Cox regression analysis will be performed to identify independent prognostic factors for graft function. STUDY END POINTS Primary End point Development of sepsis, rejection, CMV infection Death Secondary End points Post LT complications requiring surgical interventions Retransplantation Expected outcome of the project: This will help us in designing simple diagnostic tools based on microbial profiling or immune cells signatures for diagnosis of graft dysfunction and sepsis. This will also give us insight about mechanism of immune pathogenesis during the development of severe conditions and this will pave way for newer therapeutic options. Ethical issues in the study and plans to address these issues - None

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