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Development and application of the quantitative real time PCR for Epstein-Barr virus and Human Cytomegalovirus in samples of glomerulopathies and kidney transplant pediatric patients

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Author(s):
Lucas Lopes Leon
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
Defense date:
Examining board members:
Sandra Helena Alves Bonon; Lilian Monteiro Pereira Palma; Mario Luis Garcia de Figueiredo
Advisor: Sandra Helena Alves Bonon
Abstract

Viral infections are directly associated with high morbidity and mortality in immunocompromised patients, especially those transplanted and those under immunosuppressive treatments. Pediatric patients, renal transplant with glomerulopathies, are patients who are predisposed to opportunistic infections, acute rejection and graft damage due to the use of immunosuppressant, and these complications can be avoided with surveillance of these infections. In this scenario, pediatric patients are at high risk of developing diseases related to the infection due to immunological immaturity. Human cytomegalovirus (HCMV) is the most common viral pathogen associated with this group of patients, although the Epstein-Barr virus (EBV) also acts in more complicated prognoses. In this study, we verified the occurrence of active infection caused by EBV and HCMV in patients with glomerulopathies and renal transplants through viral load determined by the quantitative PCR technique (qPCR). For this, it was used urine and plasma samples from 47 pediatric patients, 37 (79%) with glomerulopathies and 10 (21%) renal transplants, analyzed during a median of 266 days for the detection of EBV and HCMV viruses. The analysis of viral genome detection in the samples was performed by the Nested-PCR technique and the detection and viral load determination for both viruses was performed by the qPCR, standardized and optimized in this study for this purpose. ln total, 623 samples were analyzed. The LMP-2A and UL83 genes which belong to EBV and HCMV were chosen, respectively, as conserved regions of the viruses, for primer and probe design and standardization of qPCR. The EBV genome was detected in 17/47 (36.2%) patients, 5/47 in transplanted patients (10.5%) and 12/47 (25.5%) with glomerulopathies. HCMV was detected in 28/47 (59.6%) patients, 7/47 in transplanted patients (15%) and 21/47 (45%) with glomerulopathies. Considering both techniques, 27 samples were EBV DNA positive and 104 were HCMV positive. The test standardized obtained a limit of detection of 83 copies/mL for EBV and 91 copies/mL for HCMV, with an incidence of 22 samples for EBV and 94 samples for HCMV by the qPCR technique. Standardization of a rapid and reliable method for detecting viral load is a difficult task, but it has great value in preventing the occurrence of diseases caused by infections, especially viral (AU)

FAPESP's process: 16/06596-0 - Detection impact analysis of adenovirus, polyomavirus, HHV 6 and 7 and EBV and HCMV quantitative detection as infectious etiology and causes of morbidities in patients with chronic glomerulopathy and pediatric kidney transplant
Grantee:Lucas Lopes Leon
Support type: Scholarships in Brazil - Master