Comparison of immunoperoxidase and immunofluorescence assays for pp65 cytomegalovirus antigen in immunocompromised patients

We compared the pp65 antigen detection by an in house method (immunoperoxidase assay) and by a commercial kit (immunofluorescence assay) available for cytomegalovirus infection diagnosis in immunocompromised patients. Sixty-four blood samples were analyzed in duplicate for both techniques. Eight-six percent of the samples had concordant qualitative results. The discordant results occurred more frequently in samples with low quantity of positive cells. There were no significant differences with qualitative and quantitative results of the methods.

Detecção do vírus Epstein-Barr em tonsilites recorrentes / Detection of Epstein-Barr virus in recurrent tonsillitis

As tonsilites recorrentes têm sido objeto de muitos estudos. Eventos considerados na predisposição e causa incluem a utilização errônea de antibióticos em crises agudas, alterações da microflora, mudanças estruturais nas criptas epiteliais tonsilares e infecções virais. A infecção pelo vírus Epstein-Barr (EBV) ocorre freqüentemente na infância persistindo em linfócitos de tonsilas, podendo causar tonsilites recorrentes. Pouco se conhece sobre a persistência e reativação do EBV em pacientes imunocompetentes. Alguns métodos como a hibridização in situ, a reação em cadeia da polimerase (PCR) e a imuno-histoquímica têm sido utilizados no estudo da patogenia do vírus. OBJETIVO: Para caracterizar a associação do vírus Epstein-Barr com tonsilites recorrentes examinamos a presença do EBV pela PCR e por imuno-histoquímica usando como alvo a proteína viral LMP-1. FORMA DE ESTUDO: Estudo transversal com análise de prevalência amostral. MATERIAL E MÉTODOS: Foram selecionados 24 blocos parafinados de tonsilas, provenientes do Serviço de Anatomia Patológica, removidas de crianças de 2 a 12 anos com diagnóstico de tonsilite recorrente. Resultados: O genoma do EBV foi detectado em 13 (54,1%) e a LMP-1 em 9 (37,5%) dos casos. CONCLUSÃO: As tonsilas das crianças podem ser colonizadas pelo EBV e este pode estar associado à patogenia das tonsilites recorrentes.

Recurrent tonsillitis has been the subject of frequent investigation. Misuse of antibiotic therapy in acute tonsillitis, changes to the tonsillar microflora, structural changes to the tonsillar crypts, and viral infections have been listed as predisposing or causal factors for recurrent tonsillitis. Epstein-Barr virus (EBV) infection usually occurs in early childhood and may persist in tonsillar lymphocytes, thus leading to the onset of recurrent tonsillitis. Little is known about the persistence and reactivation of EBV strains in immunocompetent patients. Methods such as in situ hybridization, polymerase chain reaction (PCR), and immunochemistry have been used to study the pathogenesis of the EBV. AIM: this study aims to characterize the association between EBV and recurrent tonsillitis by investigating the presence of EBV through PCR and immunohistochemistry, using viral protein LMP-1 as a target. STUDY DESIGN: this is a cross-sectional study with analysis of sample prevalence. MATERIALS AND METHOD: twenty-four paraffin-embedded tonsil specimens from the Pathology Service were selected. The specimens were removed from children aged between 2 and 12 years diagnosed with recurrent tonsillitis. RESULTS: EBV genome was detected in 13 (54.1%) specimens, whereas viral protein LMP-1 was found in 9 (37.5%) specimens. CONCLUSION: children's tonsils can be colonized by EBV and such colonies may be associated with the pathogenesis of recurrent tonsillitis.

Comparison of Quantitative Cytomegalovirus Real-time PCR in Whole Blood and pp65 Antigenemia Assay: Clinical Utility of CMV Real-time PCR in Hematopoietic Stem Cell Transplant Recipients

Successful preemptive therapy for cytomegalovirus (CMV) infection in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infection. Although the pp65 antigenemia assay has been widely used for this purpose, real-time quantification of CMV DNA has recently been recognized as an alternative diagnostic approach. However, the guidelines for antiviral therapy based on real-time quantitative polymerase chain reaction (RQ-PCR) have yet to be established. From November 2004 to March 2005, a total of 555 whole blood samples from 131 hematopoietic stem cell transplant (HSCT) recipients were prospectively collected. RQ-PCR was conducted using an Artus(R) CMV LC PCR kit (QIAGEN). Both qualitative and quantitative correlations were drawn between the two methods. Exposure to the antiviral agent influenced the results of the two assays. Additionally, the discrepancy was observed at low levels of antigenemia and CMV DNA load. Via ROC curve analysis, the tentative cutoff value for preemptive therapy was determined to be approximately 2x10(4) copies/mL (sensitivity, 80.0%; specificity, 50.0%) in the high risk patients, and approximately 3x10(4) copies/mL (sensitivity, 90.0%; specificity, 70.0%) in the patients at low risk for CMV disease. Further study to validate the optimal cutoff value for the initiation of preemptive therapy is currently underway.

effect of epstein-barr virus latent membrane protein-1 status on outcome of patients with classic hodgkin's lymphoma

This study determines the expression of latent membrane protein-1 [LMP- 1] in lymph nodes of patients with classic Hodgkin lymphoma [cHL] and its role in patients' survival. Specimen of 98 patients who had cHL was chosen and the samples were immunohistochemically stained with LMP-1 and Overall disease-free survival [DFS] was measured from the date of complete remission until the Relapse was confirmed by another lymph node biopsy. We investigated the expression of LMP-1 in outcome of patients with cHL. LMP-1 was detected in RS cells of cHL in twenty five out of ninety eight [25.5%] patients. LMP-1 expression was significantly more frequent in mixed cellularity, 46.7%, than nodular sclerosis, 15.4% [P= 0.005]. Patients with EBV-positive tumors had fewer DFS [22.6 vs. 25.9 mo], but the difference was not statistically significant [p=0.06]. Although in our study there is no relationship between age and LMP-1 expression. LMP-1 expression is associated with statistically different DFS in treated patients with cHL

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Rapid immunodiagnosis of active cytomegalovirus infection by pp65 antigenemia assay.

Detection of early antigen PP65 in blood leucocytes is a new method of diagnosing active cytomegalovirus (CMV) infection. Using a mixture of two monoclonal antibodies, CMV pp65 antigen was detected in blood leucocytes by a quantitative indirect immunofluorescence test within 3-5 hours after sampling. The pp65 antigen was detected in 20(29.8%) of the 67 immunocompromised patients included in this study. The level of CMV antigenemia appears to be related to the severity of CMV infection. Thus, this method may provide a rapid and sensitive approach to early diagnosis of active CMV infection in immunocompromised patients and a promising marker for initiating therapy and monitoring disease activity and its response to therapy.

Epstein-Barr virus in biopsies from patients with Hodgkin and non-Hodgkin lymphoma at the University of Puerto Rico immunohistochemistry laboratory

OBJECTIVES: We aimed to determine the Epstein-Barr Virus (EBV) presence rate in our laboratory's lymphoma tissue biopsies for comparison with that reported in literature. BACKGROUND: The presence of EBV has been established in Hodgkin lymphoma (HL), endemic Burkitt Lymphoma and some non-Hodgkin lymphomas (NHL). It has been linked to geographic, ethnic and socioeconomic factors, with a lower rate in developed countries. METHODS: We used the immunoperoxidase technique to determine the rate of the EBV LMP-1 in eighty-seven biopsies diagnosed as lymphoma. Tissue slides were stained using the Ventana Automated Slide Stainer with the DAKO EBV LMP-1 primary antibody and the results were analyzed with the SYSTAT program. RESULTS: We found an LMP-1 positive rate of 50 per cent for 22 cases of HL and 35 per cent for 63 cases of NHL. Among HL, 5 were children and 16 were adults, with LMP-1 positive rates of 60 per cent and 50 per cent respectively. Among NHL, 3 were children and 59 were adults, with equal LMP-1 positive rates of 33 per cent. The sex LMP-1 positive rates for HL were 42 per cent for 12 males and 60 per cent for 10 females. Among NHL, the sex LMP-1 positive rates were 39 per cent for 38 males and 28 per cent for 27 females. NHL was further subdivided into subtypes and LMP-1 primary antibody positive rates were reported. CONCLUSIONS: We found a similar presence rate of EBV in the HL biopsies to that of developed countries, but a similar presence rate of EBV in NHL biopsies to that of developing countries.

The Epstein Barr virus and gastric carcinoma.

The Epstein Barr Virus (EBV) is one of the viruses implicated in the pathogenesis of malignant tumors. Its association with Burkitt's Lymphoma, nasopharyngeal carcinoma and certain cases of Hodgkin's lymphoma is well known. More recently this virus has been found to be associated with gastric carcinoma. Studies on this association, in our country are lacking. We have studied 37 cases of carcinoma using immunohistochemistry for LMP1 protein. Most workers have found an expression of latency type I antigens in gastric adenocarcinomas, with only a few cases being LMP1 positive. In the 37 cases studied by us, 24.14% of cases were positive for LMP1. LMP1 is known to have oncogenic properties. In addition, it has been demonstrated earlier that the EBV particles in gastric carcinoma are monoclonal. These two factors support a pathogenetic role for EBV in gastric carcinoma.