A New Strategy Based On qPCR to Optimize Detection and Quantification of Eight Herpesviruses in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

• Published in: Blood 2012-11, Vol.120 (21), p.3044-3044
• Main Authors: Ueda, Miriam, Real, Juliana Monte, Oliveira, Paulo Guilherme A G, de Sa Moreira, Eloisa, Reis, Luiz Fernando Lima, Granato, Celso FH, Rodrigues, Celso Arrais
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.3044.3044

Abstract 3044 Human herpesviruses may cause severe complications after Hematopoietic Stem Cell Transplantation (HSCT) as interstitial pneumonia, encephalitis and post-transplantation lymphoproliferative disease (PTLD). Monitoring these viruses and providing precise, rapid and early diagnosis of related clinical diseases constitute an essential measure to improve outcomes. A prospective survey on the incidence and clinical features of herpesvirus infections after HSCT has not yet been performed in Brazilian patients. Additionally, the impact of most of these infections on the HSCT outcome is still unclear. We sought to develop a test based on real-time polymerase chain reaction (qPCR) to screen and quantify all known human herpesviruses (CMV, EBV, HSV1, HSV2, VZV, HHV6, HHV7 and HHV8) in plasma samples from patients submitted to HSCT. DNA purification from plasma samples has been performed with the QIAamp DNA Blood Mini Kit (manually) or with the QIAamp DNA Blood Mini QIAcube Kit and the QIAcube robot (Qiagen). At least two sets of primers previously described have been tested for each virus for the approach using SYBR Green in order to select for the sets with best efficiency and sensitivity. The sets of primers and TaqMan® probes for the hydrolysis approach have also been previously described. Lambda phage and a commercial internal positive control (IPC, Life Technologies) have been tested as internal controls. The viruses probes were labeled with FAM, while the IPC probe was labeled with VIC. All qPCR reactions have been performed in a 7900HT (Life Technologies). Infected cell cultures and plasma specimens with a known viral load/amplicon copy number have been used as controls. By august 2012, 824 whole blood and plasma were collected from 91 patients. Initially, we tested a screening approach based on three sets of triplex qPCR reactions (including the internal control) using SYBR Green and melting analysis. Although the test showed good linearity across 6 to 7 orders of magnitude in the log scale for most of the targets, the discrimination was poor for low-copy samples (≤ 103 copies of the target/ reaction) or complex samples (positive for more than one virus). We then chose to optimize a strategy based on the use of hydrolysis probes, the gold standard in molecular pathology. Except for EBV, which has been amplified and detected in a duplex reaction along with the IPC, the other amplicons have been screened and quantified in singleplex reactions. All