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

BACKGROUND 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. RESULTS 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 × 10² to 1.3 × 10⁸ copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³ to 2.0 × 10⁵ copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴ to 1.1 × 10⁵ copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10² to 5.6 × 10³ copies/ml in HIV-infected patients (n = 12), and 2.0 × 10² to 9.1 × 10⁴ copies/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). CONCLUSION 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.The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia

A healthy dietary pattern associates with a lower risk of a first clinical diagnosis of central nervous system demyelination

Background: The evidence associating diet and risk of multiple sclerosis is inconclusive. Objective: We investigated associations between dietary patterns and risk of a first clinical diagnosis of central nervous system demyelination, a common precursor to multiple sclerosis. Methods: We used data from the 2003–2006 Ausimmune Study, a case–control study examining environmental risk factors for a first clinical diagnosis of central nervous system demyelination, with participants matched on age, sex and study region. Using data from a food frequency questionnaire, dietary patterns were identified using principal component analysis. Conditional logistic regression models (n = 698, 252 cases, 446 controls) were adjusted for history of infectious mononucleosis, serum 25-hydroxyvitamin D concentrations, smoking, race, education, body mass index and dietary misreporting. Results: We identified two major dietary patterns – healthy (high in poultry, fish, eggs, vegetables, legumes) and Western (high in meat, full-fat dairy; low in wholegrains, nuts, fresh fruit, low-fat dairy), explaining 9.3% and 7.5% of variability in diet, respectively. A one-standard deviation increase in the healthy pattern score was associated with a 25% reduced risk of a first clinical diagnosis of central nervous system demyelination (adjusted odds ratio 0.75; 95% confidence interval 0.60, 0.94; p = 0.011). There was no statistically significant association between the Western dietary pattern and risk of a first clinical diagnosis of central nervous system demyelination. Conclusion: Following healthy eating guidelines may be beneficial for those at high risk of multiple sclerosis

A higher Mediterranean diet score, including unprocessed red meat, is associated with reduced risk of central nervous system demyelination in a case-control study of Australian adults

Background: The evidence associating diet and risk of multiple sclerosis (MS) is inconclusive.Objectives: The aim of this study was to investigate associations between a Mediterranean diet and risk of a first clinical diagnosis of central nervous system demyelination (FCD), a common precursor to MS.Methods: We used data from the 2003-2006 Ausimmune Study, an Australian multicenter, case-control study examining environmental risk factors for FCD, with participants matched on age, sex, and study region (282 cases, 558 controls; 18-59 y old; 78% female). The alternate Mediterranean diet score (aMED) was calculated based on data from a food-frequency questionnaire. We created a modified version of the aMED (aMED-Red) where ∼1 daily serving (65 g) of unprocessed red meat received 1 point. All other components remained the same as aMED. Conditional logistic regression (254 cases, 451 controls) was used to test associations between aMED and aMED-Red scores and categories and risk of FCD, adjusting for history of infectious mononucleosis, serum 25-hydroxyvitamin D concentrations, smoking, education, total energy intake, and dietary underreporting.Results: There was no statistically significant association between aMED and risk of FCD [per 1-SD increase in aMED score: adjusted odds ratio (aOR): 0.89; 95% CI: 0.75, 1.06; P = 0.181]. There was evidence of a nonlinear relation between aMED-Red and risk of FCD when a quadratic term was used (P = 0.016). Compared with the lowest category of aMED-Red, higher categories were significantly associated with reduced risk of FCD, corresponding to a 37% (aOR: 0.63; 95% CI: 0.41, 0.98; P = 0.039), 52% (aOR: 0.48; 95% CI: 0.28, 0.83; P = 0.009), and 42% (aOR: 0.58; 95% CI: 0.35, 0.96; P = 0.034) reduced risk of FCD in categories 2, 3, and 4, respectively.Conclusions: A Mediterranean diet, including unprocessed red meat, was associated with reduced risk of FCD in this Australian adult population. The addition of unprocessed red meat to a Mediterranean diet may be beneficial for those at high risk of MS

Higher fish consumption and lower risk of central nervous system demyelination

Background/Objectives: The evidence for diet as a risk factor for multiple sclerosis (MS) is inconclusive. We examined the associations between fish consumption and risk of a first clinical diagnosis of central nervous system demyelination (FCD), a common precursor to MS.Methods: The 2003-2006 Ausimmune Study was a case-control study examining environmental risk factors for FCD, with participants recruited from four regions of Australia and matched on age, sex, and study region. Dietary intake data were collected using a food frequency questionnaire. We used conditional logistic regression models to test associations between fish consumption (total, tinned, grilled, and fried) and risk of FCD (249 cases and 438 controls), adjusting for history of infectious mononucleosis, smoking, serum 25-hydroxyvitamin D concentrations, socio-economic status, omega-3 supplement use, dietary under-reporting, and total energy intake.Results: Higher total fish consumption (per 30 g/day, equivalent to two serves/week) was associated with an 18% reduced risk of FCD (AOR 0.82; 95% CI 0.70, 0.97). While we found no statistically significant associations between grilled and fried fish consumption and risk of FCD, higher tinned fish consumption (per 30 g/day) was associated with a 41% reduced risk of FCD (AOR 0.59; 95% CI 0.39, 0.89).Conclusions: Tinned fish is predominantly oily, whereas grilled and fried fish are likely to be a combination of oily and white types. Oily fish is high in vitamin D and very long chain polyunsaturated omega-3 fatty acids, both of which may be beneficial in relation to MS

Higher Mediterranean diet score is associated with longer time between relapses in Australian females with multiple sclerosis

A higher Mediterranean diet score has been associated with lower likelihood of multiple sclerosis. However, evidence regarding its association with disease activity and progression is limited. Using data from the AusLong Study, we tested longitudinal associations (over 10 years follow-up) between the alternate Mediterranean diet score (aMED) and aMED-Red (including moderate consumption of unprocessed red meat) and time between relapses and disability measured by Expanded Disability Status Scale (EDSS) (n=132; 27 males, 105 females). We used covariate-adjusted survival analysis for time between relapses, and time series mixed-effects negative binomial regression for EDSS. After adjusting for covariates, both higher aMED (aHR=0.94, 95%CI: 0.90, 0.99, p=0.009) and higher aMED-Red (aHR=0.93, 95%CI: 0.89, 0.97, p=0.001) were associated with significantly longer time between relapses in females. Whether specific dietary components of a Mediterranean diet are important in relation to relapses merits further study.Comment: Original article, Brief communication, 13 pages, 2 tables (one main table and one supplementary table

Higher non-processed red meat consumption is associated with a reduced risk of central nervous system demyelination

The evidence associating red meat consumption and risk of multiple sclerosis is inconclusive. We tested associations between red meat consumption and risk of a first clinical diagnosis of central nervous system demyelination (FCD), often presaging a diagnosis of multiple sclerosis. We used food frequency questionnaire data from the 2003-2006 Ausimmune Study, an incident, matched, case-control study examining environmental risk factors for FCD. We calculated non-processed and processed red meat density (g/1,000 kcal/day). Conditional logistic regression models (with participants matched on age, sex, and study region) were used to estimate odds ratios (ORs), 95% confidence intervals (95% CI) and p-values for associations between non-processed (n = 689, 250 cases, 439 controls) and processed (n = 683, 248 cases, 435 controls) red meat density and risk of FCD. Models were adjusted for history of infectious mononucleosis, serum 25-hydroxyvitamin D concentrations, smoking, race, education, body mass index and dietary misreporting. A one standard deviation increase in non-processed red meat density (22 g/1,000 kcal/day) was associated with a 19% reduced risk of FCD (AOR = 0.81; 95%CI 0.68, 0.97; p = 0.02). When stratified by sex, higher non-processed red meat density (per 22 g/1,000 kcal/day) was associated with a 26% reduced risk of FCD in females (n = 519; AOR = 0.74; 95%CI 0.60, 0.92; p = 0.01). There was no statistically significant association between non-processed red meat density and risk of FCD in males (n = 170). We found no statistically significant association between processed red meat density and risk of FCD. Further investigation is warranted to understand the important components of a diet that includes non-processed red meat for lower FCD risk

Omega-3 Index, fish consumption, use of fish oil supplements and first clinical diagnosis of central nervous system demyelination

Higher intakes of omega-3 polyunsaturated fatty acids (n3PUFAs) have been associated with lower MS risk. We aimed to test associations between the Omega-3 Index, blood levels of n3PUFAs, fish oil supplement use, and fish consumption with a first clinical diagnosis of CNS demyelination (FCD). Cases (n = 250) had a higher Omega-3 Index compared with a matched group of controls (n = 471) (average treatment effect (ATE)=0.31, p = 0.047, based on augmented inverse probability weighting). A higher percentage of cases than controls used fish oil supplements (cases=17% vs. controls=10%). We found that Omega-3 Index increased as time between FCD and study interview increased (e.g., at or below median (112 days), based on ATE, mean=5.30, 95% CI 5.08, 5.53; above median, mean=5.90, 95% CI 5.51, 6.30). Fish oil supplement use increased in a similar manner (at or below median (112 days), based on ATE, proportion=0.12, 95% CI 0.06, 0.18; above the median, proportion=0.21, 95% CI 0.14, 0.28). Our results suggest a behaviour change post FCD with increased use of fish oil supplements

Adherence to MRI protocol consensus guidelines in multiple sclerosis: An Australian multi-centre study

Introduction Multiple sclerosis (MS) is a debilitating disease that causes significant morbidity within a young demographic. Diagnostic guidelines for MS have evolved, and imaging has played an increasingly important role in diagnosis over the last two decades. For imaging to contribute to diagnosis in a meaningful way, it must be reproducible. Consensus guidelines for MRI in MS exist to define correct sequence type and imaging technique, but it is not clear to what extent they are followed. This study reviewed MRI studies performed on Australian individuals presenting with a first clinical diagnosis of central nervous system demyelination (FCD) for adherence to published guidelines and discussed practical implementation of MS guidelines in light of recent updates. Methods The Ausimmune study was a prospective case control study of Australian participants presenting with FCD from 2003 to 2006. Baseline cranial and spinal cord MRI studies of 226 case participants from four separate Australian regions were reviewed. MRI sequences were classified according to anatomical location, slice plane, tissue weighting and use of gadolinium-containing contrast media. Results were compared with the 2003 Consortium of Multiple Sclerosis Centres MRI protocol for the diagnosis of MS. Results The composition of core cranial MRI sequences performed varied across the 226 scans. Of the studies, 91% included sagittal fluid attenuated inversion recovery (FLAIR) sequences. Cranial axial T2-weighted, axial FLAIR and axial proton density-weighted sequences were performed in 88%, 60% and 16% (respectively) of scans. Only 25% of the studies included a T1-weighted contrast-enhanced sequence. Concordance with the guidelines in all sequences was very low (2). Conclusion Only a small number of MRI investigations performed included all of the sequences stipulated by consensus guidelines. This is likely due to poor awareness in the imaging community of the guidelines and the rationale behind certain sequences. Radiologists with a sub-speciality interest in neuroradiology should take ownership of this issue and ensure that recommended imaging guidelines are followed

Reported changes in dietary behavior following a first clinical diagnosis of central nervous system demyelination

Background/objectives: Although the current evidence is insufficient to recommend a special diet for people with multiple sclerosis (MS), dietary advice for people with MS is prolific online and in the media. This study aimed to describe dietary changes made in the year following a first clinical diagnosis of central nervous system demyelination (FCD), a common precursor to MS. Subjects/methods: We used follow-up data from the Ausimmune Study, a multicentre matched case-control study examining the environmental risk factors for a FCD. A total of 244 cases (60 male, 184 female) completed a 1-year follow-up interview, which included a question about dietary changes. We described the number and proportion (%) of participants who reported making dietary changes and the type of change made. We investigated independent predictors of making a dietary change using a multivariable logistic regression model. Results: A total of 38% (n = 92) of participants at the 1-year follow-up reported making at least one dietary change over the last year. There were no statistically significant independent associations between any participant characteristic and odds of making a dietary change. Of those who made at least one dietary change, the most common changes were increasing fruit and/or vegetable intake (27%, n = 25) and following a low-fat diet (25%, n = 23). Conclusion: A considerable proportion of the study population reported making at least one dietary change in the year following a FCD, with the majority of changes being toward a healthier diet. Further research is warranted to investigate the reasons behind any dietary changes adopted by people with a FCD or with MS, and whether making a dietary change has benefits for the progression of demyelinating diseases, e.g., to a diagnosis of MS, as well as for general health and well-being