Standardization of polymerase chain reaction in real time (qPCR) and consensus-degenerate hybrid oligonucleotide primer (CODEHOP) in monitoring of betaherpesvírus reactivation in hematopoietic stem cells transplant (HSCT) patients

Abstract

The human herpesviruses are major causes of infections in transplant patients and in immunocompromised patients in general. Among these complications are problems such as interstitial pneumonia, gastritis and encephalitis. Recent studies have found high levels of HHV-6 associated with increased mortality and graft-versus-host disease (GVHD) grades II-III and acute skin GVHD grades III and IV associated with CMV disease in transplant of hematopoietic progenitor cells. Currently, molecular methods for detection and quantification of infectious agents are being deployed in routine to minimize the consequences and avoid possible clinical manifestations of these herpesviruses, providing early treatment with appropriate antiviral therapy (preemptive). The Polymerase Chain Reaction Real-time is one of the options to be used in the monitoring of transplant patients. An advantage of this technique is the possibility of using the plasma neutropenic patients where this was often impossible to undertake the techniques of antigenemia in whole blood and as no diagnostic kits commercially available for this purpose, it is necessary to develop these techniques "in house" and CODEHOP PCR (consensus degenerate hybrid primers). Thus, the main objective of the study is to standardize and deploy the technique of real-time PCR, "in house" using Taqman technology and design of primers will be held from conserved viral regions and specific in the detection and quantification of DNA studied betaherpesvírus (HCMV HHV-6 and HHV-7). Also be used, consensus primers for screening for later estimation of herpes viral load. The analysis of the clinical and laboratory results obtained will be held in relation to mortality, reactivation of CMV and the occurrence of GVHD among other variables. Will be collected, weekly, plasma samples from 60 patients HSCT from the day of transplantation until 100 days after transplant. The HCMV antigenemia technique is be done. The qPCR technique is developed and used to detect and quantify the viral load of these samples. In these respects, after the project ended, we routinely use these techniques, which will assist in the care of patients with the diagnosis of these viruses. (AU)