Adam J. Branscum

Sclerostin and Dickkopf-1 in Renal Osteodystrophy

BACKGROUND AND OBJECTIVES The serum proteins sclerostin and Dickkopf-1 (Dkk-1) are soluble inhibitors of canonical wnt signaling and were recently identified as components of parathyroid hormone (PTH) signal transduction. This study investigated the associations between sclerostin and Dkk-1 with histomorphometric parameters of bone turnover, mineralization, and volume in stage 5 chronic kidney disease patients on dialysis (CKD-5D). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a cross-sectional study, 60 CKD-5D patients underwent bone biopsies followed by histomorphometry. Levels of sclerostin, Dkk-1, and intact PTH (iPTH) were determined in blood. RESULTS Serum levels of sclerostin and iPTH correlated negatively. In unadjusted analyses, sclerostin correlated negatively with histomorphometric parameters of turnover, osteoblastic number, and function. In adjusted analyses, sclerostin remained a strong predictor of parameters of bone turnover and osteoblast number. An observed correlation between sclerostin and cancellous bone volume was lost in regression analyses. Sclerostin was superior to iPTH for the positive prediction of high bone turnover and number of osteoblasts. In contrast, iPTH was superior to sclerostin for the negative prediction for high bone turnover and had similar predictive values than sclerostin for the number of osteoblasts. Serum levels of Dkk-1 did not correlate with iPTH or with any histomorphometric parameter. CONCLUSIONS Our data describe a promising role for serum measurements of sclerostin in addition to iPTH in the diagnosis of high bone turnover in CKD-5D patients, whereas measurements of Dkk-1 do not seem to be useful for this purpose.

Low Bone Volume—A Risk Factor for Coronary Calcifications in Hemodialysis Patients

BACKGROUND AND OBJECTIVES There is increasing evidence that altered bone metabolism is associated with cardiovascular calcifications in patients with stage 5 chronic kidney disease on hemodialysis (HD). This study was conducted to evaluate the association between bone volume, turnover, and coronary calcifications in HD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a cross-sectional study, bone biopsies and multislice computed tomography were performed in 38 HD patients. Bone volume/total volume, activation frequency, and bone formation rate/bone surface were determined by histomorphometry and coronary calcifications were quantified by Agatston scores. RESULTS Prevalence of low bone turnover was 50% and of low bone volume was 16%. Among the studied traditional cardiovascular risk factors, only age was found to be associated with coronary calcifications. Lower bone volume was a significant risk factor for coronary calcifications during early years of HD, whereas this effect was not observed in patients with dialysis duration >6 yr. Histomorphometric parameters of bone turnover were not associated with coronary calcifications. CONCLUSIONS Low bone volume is associated with increased coronary calcifications in patients on HD.

HIV–malaria co-infection: effects of malaria on the prevalence of HIV in East sub-Saharan Africa

OBJECTIVE To examine the association between malaria and HIV prevalence in East sub-Saharan Africa. METHODS Using large nationally representative samples of 19,735 sexually active adults from the 2003-04 HIV/AIDS indicator surveys conducted in Kenya, Malawi and Tanzania, and the atlas malaria project, we analysed the relationship between malaria and HIV prevalence adjusting for important socioeconomic and biological cofactors. RESULTS In adjusted models, individuals who live in areas with high Plasmodium falciparum parasite rate (PfPR > 0.42) had increased estimated odds of being HIV positive than individuals who live in areas with low P. falciparum parasite rate (PfPR ≤ 0.10) [men: estimated odds ratio (OR) 2.24, 95% confidence interval (CI) 1.62-3.12; women: estimated OR 2.44, 95% CI 1.85-3.21]. CONCLUSION This is the first study to report malaria as a risk factor of concurrent HIV infection at the population level. According to our results, individuals who live in areas with high P. falciparum parasite rate have about twice the risk of being HIV positive compared with individuals who live in areas with low P. falciparum parasite rate. Our work emphasizes the need for field studies focused on quantifying the interaction among parasitic infections and risk of HIV infection, and studies to explore the impact of control interventions. Programmes focused on reducing malaria transmission will be important to address, especially in HIV-infected individuals.

The Increased Prevalence of Neuropathogenic Strains of EHV-1 in Equine Abortions

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

Frequentist and Bayesian approaches to prevalence estimation using examples from Johne's disease

Abstract Although frequentist approaches to prevalence estimation are simple to apply, there are circumstances where it is difficult to satisfy assumptions of asymptotic normality and nonsensical point estimates (greater than 1 or less than 0) may result. This is particularly true when sample sizes are small, test prevalences are low and imperfect sensitivity and specificity of diagnostic tests need to be incorporated into calculations of true prevalence. Bayesian approaches offer several advantages including direct computation of range-respecting interval estimates (e.g. intervals between 0 and 1 for prevalence) without the requirement of transformations or large-sample approximations. They also allow direct probabilistic interpretation, and the flexibility to model in a straightforward manner the probability of zero prevalence. In this review, we present frequentist and Bayesian methods for animal- and herd-level true prevalence estimation based on individual and pooled samples. We provide statistical methods for detecting differences between population prevalence and frequentist methods for sample size and power calculations. All examples are motivated using Mycobacterium avium subspecies paratuberculosis infection and we provide WinBUGS code for all examples of Bayesian estimation.

Within-Subject Variability in Repeated Measures of Salivary Analytes in Healthy Adults

BACKGROUND Saliva contains a large number of biomolecules, some of which have putative diagnostic usefulness. A potential problem with the use of biomolecules in diagnosis is day-to-day fluctuation due to within-subject variability. This study evaluated the intraindividual variability of six salivary analytes in healthy adults and determined their normal range. METHODS Unstimulated whole saliva (5 ml) was collected every 2 to 3 days on six occasions from 30 subjects in good oral and systemic health. Four of the samples were collected in the clinic, and two were collected by the subject at home. The concentration ranges of interleukin (IL)-1beta, IL-6, matrix metalloproteinase-8, prostaglandin E(2), tumor necrosis factor-alpha, interferon-alpha, and albumin were examined. Descriptive statistics were computed, and a one-way random-effects model was used to quantify within- and between-subject components of variability. Intraclass correlation coefficients (ICCs) were calculated for each subject/analyte combination. RESULTS Within-subject coefficients of variation for these analytes ranged from 67.6% to 172.1% for the in-clinic samples and from 111.9% to 201.0% for the at-home samples. The ICC for the various analytes ranged from 41% to 61% for the in-clinic samples. The at-home samples exhibited significantly more variability than did those obtained in the clinic under supervision. CONCLUSIONS There was marked within-subject variation in the salivary concentrations of these analytes. With increased interest in salivary diagnostics, the within-subject variability, normal range, and threshold levels for abnormal levels of individual salivary analytes need to be determined if these diagnostics tests are to have clinical usefulness.

Development and Evaluation of One-Step TaqMan Real-Time Reverse Transcription-PCR Assays Targeting Nucleoprotein, Matrix, and Hemagglutinin Genes of Equine Influenza Virus

ABSTRACT The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

The link between bone and coronary calcifications in CKD-5 patients on haemodialysis

BACKGROUND Vascular calcifications are frequent in Stage 5 chronic kidney disease (CKD-5) patients receiving haemodialysis. The current study was designed to evaluate the associations between bone turnover/volume and coronary artery calcifications (CAC). METHODS In 207 CKD-5 patients, bone biopsies, multislice computed tomography of the coronary arteries and blood drawings for relevant biochemical parameters were done. The large number of CKD-5 patients enrolled allowed separate evaluation of patients with CAC versus patients without CAC and adjustment for traditional and non-traditional risk factors for CAC. RESULTS When all patients were analysed, associations were found between CAC and bone turnover, bone volume, age, gender and dialysis vintage. When only patients with CAC were included, there was a U-shaped relationship between CAC and bone turnover, whilst the association with bone volume was lost. In these patients, the relationship of CAC with age, gender and dialysis vintage remained. CONCLUSIONS Beyond the non-modifiable risk factors of age, gender and dialysis vintage, these data show that bone abnormalities of renal osteodystrophy amenable to treatment should be considered in the management of patients with CAC.

Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.

Intact PTH Combined With the PTH Ratio for Diagnosis of Bone Turnover in Dialysis Patients: A Diagnostic Test Study

BACKGROUND Determination of parathyroid hormone (PTH) level is the most commonly used surrogate marker for bone turnover in patients with stage 5 chronic kidney disease on dialysis therapy (CKD-5D). The objective of this study is to evaluate the predictive value of various PTH measurements for identifying low or high bone turnover rate. STUDY DESIGN Diagnostic test study. SETTINGS & PARTICIPANTS 141 patients with CKD-5D from 15 US hemodialysis centers. INDEX TESTS Intact PTH, PTH 1-84, and PTH ratio (ratio of level of PTH 1-84 to level of large carboxy-terminal PTH fragments). REFERENCE TEST OR OUTCOME Bone turnover determined using bone histomorphometry. OTHER MEASUREMENTS Demographic and treatment-related factors, serum calcium and phosphorus. RESULTS Patients presented histologically with a broad range of bone turnover abnormalities. In white patients with CKD-5D (n = 70), PTH ratio <1.0 added to intact PTH level <420 pg/mL increased the positive predictive value for low bone turnover from 74% to 90%. In black patients (n = 71), adding PTH ratio <1.2 to intact PTH level <340 pg/mL increased the positive predictive value for low bone turnover from 48% to 90%. Adding PTH ratio >1.6 to intact PTH level of 340-790 pg/mL increased the positive predictive value for high bone turnover from 56% to 71%. LIMITATIONS Because the research protocol called for carefully controlled blood specimen handling, blood drawing and routine specimen handling might be less stringent in clinical practice. By limiting study participation to black and white patients with CKD-5D, we cannot comment on the roles of intact PTH, PTH 1-84, and PTH ratio in other racial/ethnic groups. CONCLUSION In black patients with CKD-5D, the addition of PTH ratio to intact PTH measurements is helpful for diagnosing low and high bone turnover. In white patients with CKD-5D, it aids in the diagnosis of low bone turnover.