A semiquantitative PCR method (SQ-PCR) to measure Epstein-Barr virus (EBV) load: its application in transplant patients

Fil: Fellner, María Dolores. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Fil: Durand, Karina. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Fil: Correa, Mariel. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Fil: Bes, David. Hospital Nacional de Pediatría "Prof. Juan P. Garrahan"; Buenos Aires, Argentina.Fil: Alonio, Lidia V. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Fil: Teyssié, Angélica R. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Fil: Picconi, María Alejandra. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Enfermedades Infecciosas (INEI). Servicio Virus Oncogénicos; Argentina.Background: High Epstein-Barr virus load has been related to an increased risk of Posttransplant Lymphoproliferative Disorders (PTLD) in transplant recipients. Objectives: Development of a method to quantitate EBV DNA levels in peripheral blood mononuclear cells (PBMC) and evaluate its usefulness in transplant patients. Study design: We designed a semiquantitative nested PCR based on a limiting dilution analysis to detect high viral loads in PBMC. This method was applied to 25 healthy carriers, and 85 solid organ transplant recipients as follows: (A) 53 asymptomatic patients; (B) 24 symptomatic patients; (C) eight patients with PTLD. Results: In healthy carriers the reciprocal of the limiting dilution (RLD) ranged between non-detected (ND) and 1, the median RLD was ND, which is equivalent to a viral load of <1 copy per 10(5) PBMC. In the transplant population the medians RLD (range) were: (A) asymptomatic group: ND (ND-64), median equivalent to a viral load of <1 copy per 10(5) PBMC; (B) symptomatic group: 4 (ND-256), median equivalent to a range of viral load of 4-64 copies per 10(5) PBMC. (C) PTLD group: 256 (16-16384), median equivalent to a range of viral load of 256-4096 copies per 10(5) PBMC. Statistically significant differences were found between all groups: A+B vs. C (P<0.0001); A vs. B (P<0.0001); A vs. C (P<0.0001), B vs. C (P<0.0001). We also observed a good correlation between viral loads and clinical findings in four follow-up patients. Considering the RLD=256 as a cutoff point to detect transplant patients with PTLD, resulted in sensitivity 75%, specificity 96.7%, positive predictive value 60%, negative predictive value 98.3%. Conclusion: This SQ-PCR method enables us to differentiate between transplant patients with and without PTLD; therefore, it could be applied as a marker for early detection of this pathology

Observable Metabolites and Metabolomic Sampling Protocols for Managed African Savanna Elephant (Loxodonta africana) Whole Blood Using H-NMR Spectroscopy

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolomics of heparinized whole blood drawn from six African savanna elephants (Loxodonta africana) maintained on a well characterized diet. Whole blood samples obtained under behavioral restraint, then quickly frozen in liquid nitrogen, were stored at &minus;80 &deg;C until analysis. Frozen samples were thawed under controlled conditions and extracted with methanol and chloroform to separate the polar and non-polar metabolites. We identified 18 polar metabolites and 14 non-polar lipids using one-dimensional (1D) and two-dimensional (2D) NMR spectra. Despite unexpected rouleaux formation in the thawed frozen samples, spectra were consistent among animals and did not vary dramatically with age or the sex of the animal