Post-transplant lymphoproliferative disorders: From epidemiology to pathogenesis-driven treatment

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Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p

Plasma Apolipoprotein Levels Are Associated with Cognitive Status and Decline in a Community Cohort of Older Individuals

<div><h3>Objectives</h3><p>Apolipoproteins have recently been implicated in the etiology of Alzheimer’s disease (AD). In particular, Apolipoprotein J (ApoJ or clusterin) has been proposed as a biomarker of the disease at the pre-dementia stage. We examined a group of apolipoproteins, including ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH and ApoJ, in the plasma of a longitudinal community based cohort.</p> <h3>Methods</h3><p>664 subjects (257 with Mild Cognitive Impairment [MCI] and 407 with normal cognition), mean age 78 years, from the Sydney Memory and Aging Study (MAS) were followed up over two years. Plasma apolipoprotein levels at baseline (Wave 1) were measured using a multiplex bead fluorescence immunoassay technique.</p> <h3>Results</h3><p>At Wave 1, MCI subjects had lower levels of ApoA1, ApoA2 and ApoH, and higher levels of ApoE and ApoJ, and a higher ApoB/ApoA1 ratio. Carriers of the apolipoprotein E ε4 allele had significantly lower levels of plasma ApoE, ApoC3 and ApoH and a significantly higher level of ApoB. Global cognitive scores were correlated positively with ApoH and negatively with ApoJ levels. ApoJ and ApoE levels were correlated negatively with grey matter volume and positively with cerebrospinal fluid (CSF) volume on MRI. Lower ApoA1, ApoA2 and ApoH levels, and higher ApoB/ApoA1 ratio, increased the risk of cognitive decline over two years in cognitively normal individuals. ApoA1 was the most significant predictor of decline. These associations remained after statistically controlling for lipid profile. Higher ApoJ levels predicted white matter atrophy over two years.</p> <h3>Conclusions</h3><p>Elderly individuals with MCI have abnormal apolipoprotein levels, which are related to cognitive function and volumetric MRI measures cross-sectionally and are predictive of cognitive impairment in cognitively normal subjects. ApoA1, ApoH and ApoJ are potential plasma biomarkers of cognitive decline in non-demented elderly individuals.</p> </div

Opportunistic viral infection after paediatric transplantation

Background: Opportunistic viral infections can cause considerable morbidity and mortality in organ and stem cell transplanted (SCT) patients, mainly due to iatrogenic T cell dysfunction. Whereas in SCT patients, in general the immunosuppressive treatment can be discontinued after 6-12 months, for the majority of organ transplanted patients, the need for treatment is life-long. Aims: This thesis focuses on infections with cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV) and human herpes virus type 6 (HHV-6) in liver and stem cell transplanted children and on the value of quantitative viral DNA measurements. The aims were to (i) investigate the incidence and clinical picture of CMV and EBV infection the first year after liver transplantation and the usefulness of DNA quantification for identifying these infections, (ii) to compare serum and whole blood as material for the analyses and (iii) to describe infections with CMV, EBV, AdV and HHV-6 and their risk factors, identification and outcome in SCT patients during the first 6-12 months after transplantation. Methods: Serum samples, drawn the first year after transplantation from 18 liver transplanted children were retrospectively investigated for CMV DNA by a quantitative PCR from Roche (CA Monitor), and from 24 liver transplanted children for EBV DNA by real time TaqMan PCR. In the comparison of sample materials, clinical samples (10,641 for CMV and 2,855 for EBV) drawn mainly from transplant patients, were surveyed as regards to viral DNA levels in whole blood and serum. In the study of SCT children, serum samples from 47 consecutively transplanted children were retrospectively investigated with analysis of viral DNA by TaqMan PCR and related to risk factors in a multivariate analysis. Results: Any CMV marker was found in 83 % of the liver transplanted patients. Symptomatic infection was found in 22% and was associated with significantly higher CMV DNA levels (paper I). More than half of the liver transplanted patients in paper II were EBV naïve at transplantation, probably due to low median age, but 92 % had markers of EBV infection within 1 year. Symptomatic infection was found in 21%: 3 patients with post transplantation lymphoproliferative disease (PTLD) and 2 with hepatitis. In these 5 patients, the EBV DNA levels were significantly higher than in the patients with asymptomatic infection. In paper III, CMV DNA levels were only 0.2 log higher in WB as compared to serum, while EBV DNA levels were 1.5 log higher in WB than in serum. Out of 47 SCT children (paper IV), 47% developed CMV DNAaemia, 19% at levels > 104 Geq/mL, and 45% developed EBV DNAaemia, but only 6 % > 104 Geq/mL. CMV DNAaemia did not develop if neither donor nor recipient had CMV IgG. ATG and total body irradiation were independent risk factors for high CMV and EBV DNA levels. HHV-6 DNA and AdV DNA were each present in 28% of the SCT patients, in the majority in low or moderate levels. Three children died from CMV, EBV and AdV complications, representing 21 % of the total mortality after SCT, and all these 3 cases were retrospectively found to have very high viral DNA levels in serum. Conclusion: Quantification of viral DNA levels contributes to a better basis of understanding of post transplant viral infections, and is critical for taking the right actions in terms of balancing immunosuppression and antiviral measures. As sample material, serum and whole blood seemed equally useful for CMV, while for EBV whole blood was more sensitive but less specific

Opportunistic virus DNA levels after pediatric stem cell transplantation: serostatus matching, anti-thymocyte globulin, and total body irradiation are additive risk factors

Viral opportunistic infections remain a threat to survival after stem cell transplantation (SCT). We retrospectively investigated infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6), or adenovirus (AdV) during the first 6-12 months after pediatric SCT. Serum samples from 47 consecutive patients were analyzed by quantitative real-time polymerase chain reaction assay. DNAemia at any time point occurred for CMV in 47%, for EBV in 45%, for HHV6 in 28%, and for AdV in 28%. Three patients (6.3%) died of CMV-, EBV-, or AdV-related complications 4, 9, and 24 weeks after SCT, respectively, representing 21% of total mortality. These 3 cases were clearly distinguishable by DNAemia increasing to high levels. Serum positivity for CMV immunoglobulin G in either recipient or donor at the time of SCT, total body irradiation, and anti-thymocyte globulin conditioning were independent risk factors for high CMV or EBV DNA levels. We conclude that DNAemia levels help to distinguish significant viral infections, and that surveillance and prophylactic measures should be focused on patients with risk factors in whom viral complications rapidly can become fatal

Opportunistic virus DNA levels after pediatric stem cell transplantation: serostatus matching, anti-thymocyte globulin, and total body irradiation are additive risk factors

Viral opportunistic infections remain a threat to survival after stem cell transplantation (SCT). We retrospectively investigated infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6), or adenovirus (AdV) during the first 6-12 months after pediatric SCT. Serum samples from 47 consecutive patients were analyzed by quantitative real-time polymerase chain reaction assay. DNAemia at any time point occurred for CMV in 47%, for EBV in 45%, for HHV6 in 28%, and for AdV in 28%. Three patients (6.3%) died of CMV-, EBV-, or AdV-related complications 4, 9, and 24 weeks after SCT, respectively, representing 21% of total mortality. These 3 cases were clearly distinguishable by DNAemia increasing to high levels. Serum positivity for CMV immunoglobulin G in either recipient or donor at the time of SCT, total body irradiation, and anti-thymocyte globulin conditioning were independent risk factors for high CMV or EBV DNA levels. We conclude that DNAemia levels help to distinguish significant viral infections, and that surveillance and prophylactic measures should be focused on patients with risk factors in whom viral complications rapidly can become fatal

Strengthening paediatric competencies and educational skills among general paediatricians.

AIM: To develop a coherent programme that addresses the need for continuing professional development in general paediatrics and educational skills for senior paediatricians at outpatient clinics in Sweden today. METHODS: Educational needs in the target group were investigated using a questionnaire. Themes and sub-themes extracted from responses informed the curriculum formulation of the continuing professional development (CPD) programme, which was completed using a variety of learning, assessment and evaluation methods. RESULTS: Forty-six paediatricians identified 355 clinical situations. Competencies in general paediatrics and educational skills were incorporated in a CPD programme, implemented in western Sweden between 13 October 2016 and 23 May 2019, with 23 learning modules and 18 participants (male/female 3/15, median age 55 years). The participants' evaluation emphasised the importance of adult learning principles. Their responses to open reflective questions on 23 May 2109 suggested that the programme offered a learning environment in which they could develop their paediatric and educational practices and improve their mentorship, networks and work-based learning environment. They also described an enhanced feeling of joy at work. CONCLUSION: The involvement of experienced paediatricians in the programme formulation may be inspirational to clinicians and contribute to the definition, revitalisation and prioritisation of general paediatrics in Sweden in the future.This study was funded by the Healthcare Board, Region Västra Götaland</p