Epstein-Barr virus load in transplant patients: Early detection of post-transplant lymphoproliferative disorders / Carga de virus Epstein-Barr en pacientes trasplantados: detección temprana de desórdenes linfoproliferativos postrasplante

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection. Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n = 58) and without (n = 47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n = 6) and without (n = 6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1-4), advanced PTLD (categories 2-4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5 log units. The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47 log EBV gEq/10(5) PBMC or 2.30; 2.60; 4.47 log gEq/10(5) OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma. The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

La carga alta del virus Epstein-Barr se utiliza como un marcador de desórdenes linfoproliferativos postrasplante (post-transplant lymphoproliferative disorders [PTLD]). El objetivo de este estudio fue validar clínicamente un ensayo de cuantificación del virus Epstein-Barr para la detección temprana de PTLD. Se efectuó un estudio transversal en el que se analizaron muestras pareadas de células mononucleares periféricas (CMP), de plasma y de tejido linfoide orofaríngeo de niños con trasplante de órgano sólido, con PTLD (n = 58) y sin PTLD (n = 47). En el seguimiento retrospectivo se incluyeron 71 muestras pareadas de CMP y de plasma de trasplantados, con PTLD (n = 6) y sin PTLD (n = 6). La carga viral se determinó por PCR en tiempo real. Se estimó la capacidad diagnóstica para detectar PTLD (categorías: todas vs. avanzadas vs. neoplásicas) analizando diferentes valores de corte o una variación de carga mayor de 0,5 logaritmos. El mayor desempeño diagnóstico para identificar todos los PTLD, los avanzados y los neoplásicos, se obtuvo con valores de corte de 1,08; 1,60 y 2,47 log copias/10(5) en CMP y de 2,30; 2,60 y 4,48 log copias/10(5) en células de tejido linfoide orofaríngeo, respectivamente. La detección del ADN del virus Epstein-Barr en el plasma mostró una especificidad alta, pero una sensibilidad baja (todas las categorías) o alta (categorías avanzadas o neoplásicas) para identificar PTLD. Se observó el desempeño diagnóstico más alto en las siguientes condiciones: 1) al identificar una variación de carga en CMP o en plasma; 2) combinando la medición de la carga viral en CMP y en plasma. La mejor capacidad diagnóstica para identificar las etapas tempranas de los PTLD se logró mediante el seguimiento simultáneo de la carga viral en CMP y en plasma; se propone un algoritmo.

Management of post-transplant Epstein-Barr virus-related lymphoproliferative disease in solid organ and hematopoietic stem cell recipients

Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) is one of the most serious complications associated with solid organ and hematopoietic stem cell transplantation. PTLD is most frequently seen with primary EBV infection post-transplant, a common scenario for pediatric solid organ recipients. Risk factors for infection or reactivation of EBV following solid organ transplant are stronger immunosuppressive therapy regimens, and being seronegative for receptor. For hematopoietic stem cell transplantation, the risk factors relate to the type of transplant, human leukocyte antigen disparity, the use of stronger immunosuppressants, T-cell depletion, and severe graft-versus-host disease. Mortality is high, and most frequent in patients who develop PTLD in the first six months post-transplant. The primary goal of this article is to provide an overview of the clinical manifestations, diagnosis, accepted therapies, and management of EBV infection in transplant recipients, and to suggest that the adoption of monitoring protocols could contribute to a reduction in related complications.

Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection

INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 107-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p < 0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p < 0.05). In BD, PBMC and plasma, EBV loads were undetectable. CONCLUSIONS: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays. .

Virus como inductores de neoplasias cutáneas / Viruses as agents inducing cutaneous neoplasms

El rol oncogénico de los virus en las neoplasias cutáneas es conocido por el hombre desde hace más de un siglo, cuando se atribuía el origen de la verruga vulgar al virus papiloma humano (VPH). En la actualidad, las neoplasias inducidas por virus pueden agruparse en tumores sólidos y procesos linfoproliferativos. Destacan entre los primeros el VPH, del cual ahora conocemos numerosos serotipos, cada uno vinculado a una neoplasia específica, el herpesvirus humano tipo 8 que produce el sarcoma de Kaposi y el poliomavirus vinculado al carcinoma de Merkel. Entre los procesos linfoproliferativos debemos mencionar al virus linfotrópico de células T humanas tipo 1 (HTLV-1) responsable de los linfomas de células T, en los cuales el compromiso cutáneo es inespecífico, con un amplio espectro de presentaciones clínicas y, que por consiguiente, plantean un reto para el diagnóstico diferencial. En este grupo también se encuentra el virus Epstein Barr vinculado a los linfomas nasales de Células NK/T y a los linfomas tipo Hidroa, de reciente descripción. En esta era en la que lo genético y lo molecular priman en las investigaciones en cáncer, no podemos dejar de lado el concepto de neoplasia como resultado de la infección por un agente viral, lo que abre una nueva veta de posibilidades de tratamiento anticanceroso basado en medicamentos antivirales.

The oncogenic role of viruses in cutaneous neoplasms has been known by humankind for more than a century, when the origin of the common wart, or verruca vulgaris, was attributed to the human papilloma virus (HPV). Currently, virus-induced cutaneous neoplasms may be grouped into solid tumors and lymphoproliferative disorders. HPV, from which various serotypes are now known, each being linked to a specific neoplasm, the human herpes virus type 8 producing Kaposi sarcoma, and the Merkel cell polyomavirus, highlight among the first group. Regarding the lymphoproliferative disorders, we should mention the human T-lymphotropic virus type I (HTLV-1), which is responsible for the T-cell lymphomas, in which the cutaneous manifestations are non-specific and have a wide spectrum, thus posing a challenge for differential diagnosis. The Epstein Barr virus, linked to nasal lymphomas of NK/T-cells and Hydroa-like cutaneous lymphomas, is also part of this group. In an era in which the genetic and molecular aspects of cancer research prevail, we may not leave behind the concept of neoplasms as a result an infection with a viral agent, which opens a wide array of new possibilities for cancer treatment based on antiviral drugs.

Profilaxis de enfermedad por virus de Epstein Barr en niños y adultos receptores de trasplante de órganos sólidos y de precursores hematopoyéticos / Prophylaxis against Epstein Barr disease in pediatric and adult patients undergoing solid organ and hematopoietic stem cells transplantation

Post transplant lymphoproliferative disease (PTLD) associated with EBV infection is one of the most life-threatening complications in SOT and HSCT. Risk factors for infection or reactivation of EBV in SOT are the use of greater immunosuppression, seronegative receptor and CMV infection. In HSCT, the risk factors are related to type of transplant, HLA disparity, the greater immunosuppression, T-cell depletion and severe GVHD. There is no scientific evidence to support the use of specific therapy for prophylaxis of EBV infection. Prophylaxis recommendations focus on avoid exposure of transplant recipients to sources of virus, through hygiene practices such as hand washing (A3), avoid sharing utensils (B3) and avoid contact with potentially infected secretions (respiratory or saliva) (A2). For PTLD prevention, the recommendation is regular EBV viral load monitoring by rtPCR. In SOT with logarithmic rising of EBV loads, it is recommended to reduce immunosuppression and periodically perform exams to diagnose PTLD. In HSCT, it is recommended to reduce immunosuppression whenever possible, and use rituximab according to speciic protocol. Acyclovir or gancyclovir have not proven to be of any eficacy in PTLD prophylaxis in SOT (C3) or HSCT (D2), so their administration as preemptive therapy is no recommended.

El síndrome linfoproliferativo (SLP) asociado a VEB constituye una grave complicación en TOS y en TPH. Los factores de riesgo de infección o reactivación de VEB en TOS son el uso de mayor inmunosupresión, la seronegatividad del receptor previa al trasplante y la infección por CMV. En TPH se consideran factores de riesgo el tipo de trasplante, disparidad HLA, mayor inmunosupresión, depleción linfocitaria y enfermedad injerto contra hospedero (EICH) grave. No hay evidencia cientíica que apoye el uso de medidas especíicas de proilaxis en prevención de infección por VEB. Se recomienda evitar la exposición a fuentes del virus de los candidatos a trasplantes a través de prácticas de higiene tales como lavado de manos (A3), evitar el compartir utensilios (B3) y evitar el contacto con potenciales secreciones infectadas (respiratorias o saliva) (A2). Para la prevención de SLP, se recomienda un esquema de monitoreo periódico de carga viral de VEB por RPC-TR. En el caso de TOS con cargas de VEB en ascenso logarítmico, se recomienda disminuir inmuno-supresión y buscar activa y periódicamente la aparición de SLP. En TPH, se recomienda, en lo posible, disminuir la inmunosupresión y se reserva el uso de rituximab para casos especíicos según protocolo. El uso de aciclovir o ganciclovir no han demostrado constituir medidas profilácticas efectivas en TOS (C3) ni en TPH (D2), no siendo recomendada su administración en esquemas de terapia anticipada.

Infección por el HTLV-I en pacientes con síndromes linfoproliferativos en dos sitios centinela de Cuba / Human T-cell lymphotropic virus type I infection in patients with lymphoproliferative disorders at two sentinel sites in Cuba

OBJETIVO: Determinar la frecuencia de la infección por el HTLV-I en pacientes con síndromes linfoproliferativos, así como en sus familiares y contactos sexuales, en dos sitios de vigilancia centinela en Cuba. MÉTODOS: Se analizaron todos los pacientes que tenían un diagnóstico presuntivo de neoplasias hematológicas entre enero de 1996 y enero de 2007 atendidos en los servicios de hematología del Hospital Hermanos Ameijeiras (HHA), de Ciudad de La Habana, y el Hospital Provincial Comandante Faustino Pérez (HPCFP), de Matanzas, Cuba. Se determinó la seropositividad al HTLV-I por ELISA y western blot y se confirmó la infección mediante la reacción en cadena de la polimerasa. Se estudiaron también los familiares y los contactos sexuales de los pacientes positivos. Se utilizó la prueba de la Z para la comparación de proporciones. RESULTADOS: La seroprevalencia de la infección por el HTLV-I en pacientes con síndromes linfoproliferativos fue de 0,4 por ciento, mayor en el HPCFP que en el HHA (6,1 por ciento frente a 0,2 por ciento; P < 0,001). No se encontraron diferencias significativas en la frecuencia de la infección según la edad, el sexo y el color de la piel. De los 53 familiares y contactos sexuales estudiados, 8 (15,1 por ciento) tuvieron diagnóstico positivo de infección por el HTLV-I. CONCLUSIÓN: La frecuencia de la infección por el HTLV-I en el grupo estudiado fue superior a la encontrada con anterioridad en Cuba. Se confirmó la utilidad de la vigilancia seroepidemiológica mediante centros centinela.

OBJECTIVE: To determine the prevalence of human T-cell lymphotropic virus type I (HTLV-I) infection among patients with lymphoproliferative disorders, as well as among their family members and sexual contacts, at two sentinel sites in Cuba. METHODS: An analysis was conducted of all the patients with a presumptive diagnosis of hematological malignancies seen by the hematology departments of the Hospital Hermanos Ameijeiras (HHA), City of Havana, and the Hospital Provincial Comandante Faustino Pérez (HPCFP), Matanza, Cuba, in January 1996-January 1997. HTLV-I seropositivity was determined by ELISA and Western Blot, and infection was confirmed by polymerase chain reaction. The positive patients' family members and sexual contacts were also assessed. The Z-test was used to compare proportions. RESULTS: Seroprevalence of HTLV-I infection in patients with lymphoproliferative disorders was 0.4 percent higher at the HPCFP than at the HHA (6.1 percent versus 0.2 percent, P < 0.001). There were no significant differences in prevalence by age, sex, or skin color. Of the 53 family members and sexual contacts studied, 8 (15.1 percent) were positive for HTLV-I infection. CONCLUSION: The prevalence of HTLV-I in the study group was higher than previously found in Cuba. The value of seroepidemiological surveillance through sentinel sites was confirmed.

Epstein-Barr virus nuclear antigen-2 detection and typing in immunocompromised children correlated with lymphoproliferative disorder biopsy findings

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, plays a significant role as a cofactor in the process of tumorigenesis, and has consistently been associated with a variety of malignancies especially in immunocompromised patients. Forty-four children and adolescents (21 liver transplant patients, 7 heart transplant, 5 AIDS, 3 autoimmune hepatitis, 2 nephritic syndromes, 2 medullar aplasia, 2 primary immunodeficiency disorder patients, 1 thrombocytopenic purpura and 1 systemic lupus erythematosus) presenting with chronic active EBV infection (VCA-IgM persistently positive; VCA-IgG > 20 AU/mL and positive IgG _ EBNA) had peripheral blood samples obtained during clinically characterized EBV reactivation episodes. DNA samples were amplified in order to detect and type EBV on the basis of the EBNA-2 sequence (EBNA2 protein is essential for EBV-driven immortalization of B lymphocytes). Although we have found a predominance of type 1 EBNA-2 virus (33/44; 75 percent), 10 patients (22.73 percent) carried type 2 EBNA-2, and one liver transplant patient (2.27 percent) a mixture of the two types, the higher proportion of type 2 EBV, as well as the finding of one patient bearing the two types is in agreement with other reports held on lymphoproliferative disorder (LPD) patients, which analyzed tumor biopsies. We conclude that EBNA-2 detection and typing can be performed in peripheral blood samples, and the high prevalence of type 2 in our casuistic indicates that this population is actually at risk of developing LPD, and should be monitored.