Does the 48-hour BH4 loading test miss responsive PKU patients?

BackgroundPhenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism. Besides dietary treatment, some patients are responsive to and treated with tetrahydrobiopterin (BH4). Our primary objective was to examine whether the 48-hour BH4 loading test misses BH4-responsive PKU patients. Secondary, we assessed if it would be beneficial to 1) use a cut-off value of 20% Phe reduction instead of commonly used 30%, and 2) extend the loading test to 7 days.Methods24 patients with a 20–30% decrease of blood Phe levels during their initial 48-hour BH4 loading test or at least one mutation associated with long-term BH4 responsiveness, were invited to participate. 22 of them underwent the 7-day BH4 loading test. During the BH4 loading test, BH4 was administered orally once daily for 7 days (20 mg/kg/day). Blood samples on filter paper were collected at 13 time points. Potential BH4 responders (≥20% decrease in blood Phe concentrations at ≥1 moment within the first 48 h or ≥30% at ≥1 moment during the entire test) underwent a treatment trial to assess true long-term responsiveness (≥30% decrease of Phe levels compared to baseline and/or ≥50% increase in natural protein tolerance in accordance with the Dutch guidelines before 2017). The duration of the treatment trial varied from 2 to 18 months.ResultsOf the 22 patients who completed the 7-day BH4 loading test, 2 were excluded, 8 had negative tests and 12 were considered to be potential BH4 responders. Of these 12 potential BH4-responsive PKU patients, 5 turned out to be false positive, 6 true-responder and 1 was withdrawn.ConclusionEven though the 48-hour BH4 loading test has proven its efficacy in the past, a full week may be necessary to detect all responders. So, if blood Phe concentrations during the 48-hour BH4 test shows a clear tendency, but not sufficient decrease, a full week (with only measurements each 24 h) could be offered. A threshold of ≥20% decrease within 48 h is not useful for predicting true BH4 responsiveness.     

Sapropterin therapy increases stability of blood phenylalanine levels in patients with BH4-responsive phenylketonuria (PKU)

It has recently been demonstrated that variability in blood phenylalanine levels is inversely correlated with IQ and is a better predictor of IQ in early and continuously treated patients with phenylketonuria (PKU) than mean blood phenylalanine levels. This suggests that stability of blood phenylalanine should be a therapeutic goal in patients with PKU. The purpose of this study was to determine if treatment with sapropterin in patients with BH4-responsive PKU would increase the stability of blood phenylalanine levels. The records of all patients treated with sapropterin in the PKU Clinic at Children's Memorial Hospital in Chicago were examined retrospectively. Patients were included in the study if they were responsive to sapropterin during a 2- to 4-week challenge (reduction of blood phenylalanine level of at least 25% after 2 weeks of therapy or, in the case of patients with well-controlled blood phenylalanine at the time of testing, increased dietary phenylalanine tolerance by 4 weeks of treatment). A total of 37 subjects were eligible for inclusion (16 male; 21 female); the mean age was 12.6 years (range, 1.5–32.0). The total number of observations (phenylalanine levels) for all subjects was 1391 with a mean of 39 per subject (range, 13–96). Linear mixed modeling was utilized to estimate variances of the blood phenylalanine before (pre) and after (post) starting sapropterin. Likelihood ratio test was performed using SAS 9.1. Means and standard deviations for phenylalanine as estimated by the model were 6.67 mg/dl (4.20) and post 5.16 (3.78). The mean level post-sapropterin was significantly lower (p = .0002). The within-subject variances (mean and SD) of phenylalanine were: pre 6.897 (2.62) and post 4.799 (2.19). These two variances are significantly different with a p = .0017. We conclude that sapropterin therapy results in increased stability of blood phenylalanine levels. This effect is likely to improve cognitive outcome in BH4-responsive patients with PKU.     

Correlation between HIV-1 viral load quantification in plasma,dried blood spots,and dried plasma spots using the Roche COBAS Taqman assay

BackgroundThe use of simplified methods for viral load determination could greatly increase access to treatment monitoring of HIV patients in resource-limited countries.ObjectiveThe aim of the present study was to optimize and evaluate the performance of the Roche COBAS Taqman assay in HIV-RNA quantification from dried blood spots (DBS) and dried plasma spots (DPS).Study designEDTA blood samples from 108 HIV-infected women were used to prepare 129 DBS and 76 DPS on Whatman 903 card. DBS and DPS were stored at ?20 °C. HIV-1 RNA was extracted from DBS/DPS using the MiniMAG system (bioMerieux). Amplification and detection were performed using the Roche COBAS TaqMan assay. Plasma viral load results were used as standard.ResultsThere was a high correlation between measures of viral load in plasma and in DBS/DPS (r = 0.96 and 0.85 respectively, P < 0.001). Overall, viral load values in DBS and DPS tended to be lower than in plasma with mean (SD) differences of 0.32 log (0.22) for DBS and of 0.35 (0.33) for DPS. Detection rates were 96.4% for DBS and 96.1% for DPS in samples with corresponding plasma values >3.0 log copies/ml. Samples with HIV-RNA below 50 copies/ml were correctly identified in 18/19 DBS and in 7/7 DPS.ConclusionsBoth DBS and DPS provided results highly correlated to the plasma values. High detection rate was obtained with both DBS and DPS when HIV-RNA was >3.0 log copies/ml. Our results support the use of DBS/DPS to detect virologic failure in resource-limited settings.     

Long term stability of HBsAg,anti-HBc and anti-HCV in dried blood spot samples and eluates

BackgroundDried blood spots (DBS) are a useful specimen collection tool in situations where venous access is problematic, however, detection of biomarkers from this specimen type is subject to variation depending on storage conditions and storage time.ObjectivesThe objective of this study is to assess the detection of HBsAg, anti-HBc and anti-HCV from DBS after storage.Study designDBS specimens were stored at −70 °C, −20 °C, 4 °C, 22 to 28 °C and 37 °C either with or without desiccant. Eluates were also prepared from DBS specimens and stored at −20 °C and −70 °C. DBS cards and eluates were tested for HBsAg, anti-HBc and anti-HCV at baseline on day 0 and thereafter at intervals of 14, 70 and 200 days.ResultsLoss of detection of both HBsAg and anti-HBc was evident by the first time point (14 days) in all storage conditions except for the samples (DBS and eluates) stored at −20 °C or −70 °C. Both HBsAg and anti-HBc stored under these conditions showed minimal variation up to the final time point (200 days) of storage. The detection of anti-HCV did not differ between the 22 to 28 °C, 4 °C, −20 °C and −70 °C DBS nor the −20 °C or the −70 °C stored eluates over the 200 day time period.ConclusionWe suggest that extended storage of DBS intended for downstream testing is best carried out by freezing either the DBS, or eluate, at −20 °C or −70 °C as soon as possible following collection for optimal detection of HBsAg, anti-HBc and anti-HCV.     

Hepatitis B viral load in dried blood spots: A validation study in Zambia

BackgroundAccess to hepatitis B viral load (VL) testing is poor in sub-Saharan Africa (SSA) due to economic and logistical reasons.ObjectivesTo demonstrate the feasibility of testing dried blood spots (DBS) for hepatitis B virus (HBV) VL in a laboratory in Lusaka, Zambia, and to compare HBV VLs between DBS and plasma samples.Study designPaired plasma and DBS samples from HIV-HBV co-infected Zambian adults were analyzed for HBV VL using the COBAS AmpliPrep/COBAS TaqMan HBV test (Version 2.0) and for HBV genotype by direct sequencing. We used Bland-Altman analysis to compare VLs between sample types and by genotype. Logistic regression analysis was conducted to assess the probability of an undetectable DBS result by plasma VL.ResultsAmong 68 participants, median age was 34 years, 61.8% were men, and median plasma HBV VL was 3.98 log IU/ml (interquartile range, 2.04–5.95). Among sequenced viruses, 28 were genotype A1 and 27 were genotype E. Bland–Altman plots suggested strong agreement between DBS and plasma VLs. DBS VLs were on average 1.59 log IU/ml lower than plasma with 95% limits of agreement of −2.40 to −0.83 log IU/ml. At a plasma VL ≥2,000 IU/ml, the probability of an undetectable DBS result was 1.8% (95% CI: 0.5–6.6). At plasma VL ≥20,000 IU/ml this probability reduced to 0.2% (95% CI: 0.03–1.7).ConclusionsIn a Zambian laboratory, we observed strong agreement between DBS and plasma VLs and high sensitivity in DBS at plasma VL ≥2,000 IU/ml. As HBV treatment expands, DBS could increase access to HBV VL testing and care in SSA settings.     

Neurofilament ELISA validation

BackgroundNeurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance.MethodsThe UmanDiagnostics NF-light ®enzyme-linked immunoabsorbent assays (ELISA) for the neurofilament light chain (NfL, 68 kDa) was used to test the intra-assay and inter-laboratory coefficient of variation (CV) between 35 laboratories worldwide on 15 cerebrospinal fluid (CSF) samples. Critical factors, such as sample transport and storage, analytical delays, reaction temperature and time, the laboratories' accuracy and preparation of standards were documented and used for the statistical analyses.ResultsThe intra-laboratory CV averaged 3.3% and the inter-laboratory CV 59%. The results from the test laboratories correlated with those from the reference laboratory (R = 0.60, p < 0.0001). Correcting for critical factors improved the strength of the correlation. Differences in the accuracy of standard preparation were identified as the most critical factor. Correcting for the error introduced by variation in the protein standards improved the correlation to R = 0.98, p < 0.0001 with an averaged inter-laboratory CV of 14%. The corrected overall inter-rater agreement was subtantial (0.6) according to Fleiss' multi-rater kappa and Gwet's AC1 statistics.ConclusionThis multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.     

The effects of time of venipuncture on variation of serum constituents. Consideration of within-day and day-to-day changes in a group of healthy young men.

Within-day and the day-to-day variations of serum constituents were evaluated in 11 healthy young men. Eighteen constituents, including electrolytes, metabolites, proteins, and enzymes, were assayed using the AutoChemist MultiChannel Analytic System. Venipunctures were performed at three hours of the day, 8 A.M., 11 A.M., and 2 P.M., on five separate experimental days. A three-factor analysis of variance model was employed to separate analytic variation from biological sources of variation. Statistically significant (p less than .05) group diurnal patterns (main effect of hour) during the six-hour period were found for serum lipids, iron, urea, albumin, total protein, and chloride. A unique individual diurnal pattern (subject-hour interaction) was statistically significant for serum potassium. Statistically significant main effect of month (main effect of day) for the group of subjects was seen for total lipids and potassium; however, the subject-day interaction term, which is an index of the day-to-day variation for the subjects, was significant (p less than .05) for all of the constituents except for sodium ion. The comparison of the variation expected within-day versus the variation seen day-to-day over four months was made by pooling the sources of within-day variation (main effect of hour + subject-hour interaction + subject-day-hour interaction) and by pooling the day-to-day variation terms (main effect of day + subject-month interaction). For serum cholesterol, potassium, acid phosphatase, and phosphate ion, the within-day variation was greater than the day-to-day variation occurring over four months, while the other constituents showed day-to-day variations of a greater magnitude than that experienced during the six-hour period.     

Phenylalanine and tyrosine measurements across gestation by tandem mass spectrometer on dried blood spot cards from normal pregnant women

PurposeMaternal phenylketonuria (MPKU) requires strict control of phenylalanine (Phe) and supplemental tyrosine (Tyr). Monitoring during pregnancy using dried blood spot (DBS) cards by tandem mass spectrometry (MS/MS) is now standard practice, however there are no Phe and Tyr reference ranges for DBS MS/MS method in healthy pregnant women.MethodsDBS cards (63–1364 days in storage) from healthy women with singleton pregnancies were analyzed by MS/MS. Three hundred ninety DBS cards from 170 pregnancies (5/1–39/6 weeks’ gestation), were tested.ResultsBoth Phe and Tyr levels declined from the first trimester (Phe: 36.2 +/− 10.6; Tyr 25.7 +/− 9.7 µmol/L) to the second trimester (Phe 33.4+/−9.3; Tyr 21.7+/− 6.7 µmol/L) and remained stable in the third trimester (Phe 32.3 +/− 8.7; Tyr 21.0 +/− 6.6 µmol/L). Phe and Tyr levels declined over time since collection (Phe: 0.004 µmol/L per day; Tyr 0.002 µmol/L). Nomograms by gestational age were created using raw data and data adjusted for time from sample collection. Reference ranges by trimester are provided.ConclusionsBoth Phe and Tyr decline quickly during the first trimester and remain relatively constant over the second and third trimesters. These nomograms will provide a valuable resource for care of MPKU.     

Forearm and leg amino acid metabolism in the basal state and during combined insulin and amino acid stimulation after a 3‐day fast

Aim: Fasting is characterized by a progressive loss of protein, but data on protein kinetics are unclear and few have studied the effects of re‐feeding. The present study was designed to test the hypothesis that a combined infusion of insulin and amino acids after fasting would induce compensatory increases in protein synthesis and reductions in protein breakdown at the whole body level and in muscle. Methods: We included 10 healthy male volunteers and studied them twice: (1) in the post‐absorptive state and (2) after 72 h of fasting. Amino acid kinetics was measured using labelled phenylalanine and tyrosine, whole body energy expenditure was assessed and urea nitrogen synthesis rates were calculated. Results: After fasting we observed an increase in arterial blood concentration of branched chain amino acids and a decrease in gluconeogenic amino acids (P < 0.05). Isotopically determined whole body, forearm and leg phenylalanine fluxes were unaltered apart from a 30% decrease in phenylalanine‐to‐tyrosine conversion (2.0 vs. 1.4 μmol kg^?1 h^?1, P < 0.01). During infusion of insulin and amino acids, amino acid concentrations increased. Conclusion: Our data indicate that after a 72‐h fast basal and insulin/amino acid‐stimulated regional phenylalanine fluxes in leg and forearm muscle are unaltered. During fasting concentrations of gluconeogenic amino acids decrease and hepatic and/or renal phenylalanine‐to‐tyrosine conversion decreases. Thus, as opposed to glucose and lipid metabolism, fasting does not induce insulin resistance as regards amino acid metabolism.     

ESI-MS/MS measurement of free carnitine and its precursor γ-butyrobetaine in plasma and dried blood spots from patients with organic acidurias and fatty acid oxidation disorders

BackgroundIn patients with fatty acid oxidation disorders (FAODs) and organic acidurias (OAs) “secondary carnitine deficiency” occurs. In OAs carnitine supplementation is widely performed and dose is often adjusted to blood-free carnitine levels. Dried blood spots (DBS) are mostly used to measure carnitine status, however measurements in plasma are discussed to be more accurate. The concentration and the predictive value of the carnitine precursor γ-butyrobetaine in blood during carnitine deficiency are unknown.MethodsFree carnitine and γ-butyrobetaine were quantified by tandem mass spectrometry in plasma and DBS from supplemented patients with OAs (n = 18) and unsupplemented patients with FAODs (n = 66) and were compared with healthy controls (n = 50).ResultsCarnitine concentrations in plasma were significantly higher than in DBS. In contrast, γ-butyrobetaine concentrations in plasma were significantly lower than in DBS. Supplemented patients had high free carnitine concentrations in combination with high γ-butyrobetaine concentrations. Unsupplemented carnitine palmitoyltransferase I-deficient patients had exceptionally high free carnitine concentrations without elevated γ-butyrobetaine, however, carnitine in plasma was much lower than in DBS. In patients with low carnitine, γ-butyrobetaine in plasma is no evidence of induced carnitine biosynthesis.ConclusionsParallel measurements in plasma and DBS demonstrated that numerous patients with low values in DBS had normal values when measured in plasma, suggesting plasma to be the more appropriate medium to use for carnitine status monitoring. In contrast, diagnosis of CPT-I deficiency may be missed when analysis is performed in plasma. Carnitine supplementation presumably inhibits γ-butyrobetaine dioxygenase and results in high γ-butyrobetaine.     

Testosterone implants in women: Pharmacological dosing for a physiologic effect

ObjectivesThe objectives of this study were to determine therapeutic serum testosterone (T) levels/ranges and inter-individual variance in women treated with subcutaneous T implants.Study designIn study group 1, T levels were measured at two separate time intervals in pre- and post-menopausal women treated with subcutaneous T for symptoms of androgen deficiency: (i) four weeks after pellet insertion, and (ii) when symptoms of androgen deficiency returned.In a separate pharmacokinetic study (study group 2), 12 previously untreated postmenopausal women each received a 100 mg T implant. Serum T levels were measured at baseline, 4 weeks and 16 weeks following T pellet implantation.In study ‘group’ 3, serial T levels were measured throughout a 26 h period in a treated patient.ResultsIn study group 1, serum T levels measured at ‘week 4’ (299.36 ± 107.34 ng/dl, n = 154), and when symptoms returned (171.43 ± 73.01 ng/dl, n = 261), were several-fold higher compared to levels of endogenous T. There was significant inter-individual variance in T levels at ‘week 4’ (CV 35.9%) and when symptoms returned (CV 42.6%). Even with identical dosing (study group 2), there was significant inter-individual variance in T levels at ‘week 4’ (CV 41.9%) and ‘week 16’ (CV 41.6%). In addition, there was significant intra-individual circadian variation (CV 25%).ConclusionsPharmacologic dosing of subcutaneous T, as evidenced by serum levels on therapy, is needed to produce a physiologic effect in female patients. Safety, tolerability and clinical response should guide therapy rather than a single T measurement, which is extremely variable and inherently unreliable.     

Influence of combined exercise and gravity transients and apnea on hemodynamics

Hemodynamic responses to combined heavy dynamic leg exercise (hiP), breath holding (BH) and gravity-induced blood volume shifts direction were studied. Thirteen subjects were studied at normal gravity and 12 during parabolic flight, performing 20 s hiP or combined hiP&BH (stimulus period) from a baseline of 30 W at normal gravity (1 G_z+). Heart rate and mean arterial pressure responses to BH were similar between gravity conditions, but stroke volume (SV) differed markedly between gravity conditions: at 1 G_z+ SV was higher [112 ± 16 ml (mean ± SD)] during BH, than during eupnea [101 ± 17 ml (P < 0.05, N = 13)]. In weightlessness the corresponding SV values were 105 ± 16 and 127 ± 20 ml, respectively (P < 0.05, N = 6). Transthoracic electrical conductance (TTC) was used as index for intrathoracic volume. TTC fell significantly during BH. This decrease was attenuated in weightlessness. It is concluded that the transient microgravity temporarily reduces the efficiency of the muscle pump so that the deep inspiration at the onset of the high-intensity exercise and breath-hold period cannot augment venous return as it could during identical manoeuvres at normal gravity.     

Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection

BackgroundDried blood spots (DBS) may be valuable in the diagnosis of congenital cytomegalovirus (CMV) infection. However, the 2007 European Quality Control for Molecular Diagnostics (QCMD) proficiency testing programme showed that CMV DNA detection in DBS was lacking sensitivity in a considerable number of participating laboratories.ObjectiveTo compare DNA extraction methods for DBS for detecting CMV. Sensitivity and applicability of the methods for high-throughput usage were assessed.Study designGuthrie cards were spotted with CMV DNA-positive whole blood (n = 15). DNA was extracted from the DBS using different extraction methods, followed by CMV amplification by means of real-time PCR.ResultsSignificant differences between the extraction methods with respect to the sensitivity were found. Optimal sensitivity was achieved when samples were tested in triplicate, demonstrating that the methods in general operated around their detection limits. Triplicate testing using the protocol by Barbi et al. [Barbi M, et al. Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection. J Clin Virol 2000;17:159–65], representing the most sensitive methods, resulted in sensitivities of 100%, 86%, and 50% for DBS with CMV DNA loads of 5–4, 4–3, and 3–2 log₁₀ copies/ml, respectively. This indicates that sensitivity limitations apply in the clinically relevant concentration range. Few methods appeared suitable for 96-well format high-throughput testing.DiscussionWhen considering universal neonatal screening for congenital CMV infection, an assay which is both sensitive and applicable for high-throughput testing is required. The protocol by Barbi et al. and the BioRobot Universal System appear appropriate candidates currently available for 96-well format application in neonatal screening using DBS.     

Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

BackgroundInfection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5–2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).⁷ObjectivesEvaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards.Study designWe compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol–chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR.ResultsThe highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol–chloroform method in combination with the nested-PCR and 6 mm, 3 × 3 mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR.ConclusionThese results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.     

Day-to-day variability in voluntary wheel running among genetically differentiated lines of mice that vary in activity level

This study examined the day-to-day variability in voluntary wheel-running behavior among three genetically distinct lines of young male and female mice. Daily wheel revolutions were recorded at an age of 6–8 weeks in 10 males and 10 females from each of 3 lines: selectively bred line for high wheel running (Line 8), selectively bred for high wheel-running activity and fixed for a Mendelian recessive allele that reduces hind-limb muscle mass by 50% (Line 3), non-selected control (Line 2). There were significant mean differences in revolutions/day among weeks (P = 0.003), but the effect size was small (10%). Significant main effects for wheel running were also revealed for sex and line (P < 0.001). The grand mean ± SD for the coefficient of variation (CV) of intra-individual wheel running was 23.0 ± 10.8%. Although a significant main effect for the CV was found for week, the effect size was low (7%) (age 6 weeks, 23.4 ± 10.9%; age 7 weeks, 25.1 ± 13.2%; age 8 weeks, 20.1 ± 7.8%). The overall mean CV was similar between females (21.4 ± 9.8%) and males (24.4 ± 12.0%) and among lines (Line 2, 23.4 ± 9.8%; Line 3, 20.4 ± 7.6%; and Line 8, 25.0 ± 14.4%). These findings are consistent with our previous work in young humans and lend further support for the hypothesis that biological mechanisms influence daily levels of physical activity.     

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay

ObjectiveFDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm.MethodsBRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms.ResultsBRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p = 0.0133), but fewer infections than BRC-plasma (p = 0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p = 0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens.ConclusionsThe DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture.     

Determinants of the variability of heart rate measures during a competitive period in young soccer players

Measurements of exercise heart rate (HR_ex), HR recovery (HRR) and HR variability (HRV) are used as indices of training status. However, the day-to-day variability of these indices throughout a competitive soccer period is unknown. On 14 occasions during a 3-week competition camp, 18 under 15 (U15) and 15 under 17 (U17) years soccer players performed a 5-min submaximal run, followed by a seated 5-min recovery period. HR_ex was determined during the last 30 s of exercise, while HRR and HRV were measured during the first and last 3 min of the post-exercise recovery period, respectively. U15 players displayed greater HR_ex (P = 0.02) and HRR (P = 0.004) compared with the U17 players, but there was no difference in HRV (P = 0.74). The mean coefficient of variation (CV) for HR_ex was lower than that for HRV [3.4 (90% CL, 3.1, 3.7) vs. 10.7 (9.6, 11.9)%, P < 0.001]; both were lower than that for HRR [13.3 (12.2, 14.3)%, P < 0.01]. In contrast to HR_ex and HRR, the CV for HRV was correlated to maximal aerobic speed (r = −0.52, P = 0.002). There was no correlation between total activity time (training sessions + matches) and CV of any of the quantified variables. The variability of each of these measures and player fitness levels should be considered when interpreting changes in training status.     

Dose-response effects of free fatty acids on amino acid metabolism and ureagenesis

Aim: Free fatty acids (FFAs) are important fuels and have vital protein‐sparing effects, particularly during conditions of metabolic stress and fasting. However, it is uncertain whether these beneficial effects are evident throughout the physiological range or only occur at very high FFA concentrations. It is also unclear whether secondary alterations in hormone levels and ketogenesis play a role. We therefore aimed at describing dose–response relationships between amino acid metabolism and circulating FFA concentrations at clamped hormone levels. Methods: Eight healthy men were studied on four occasions (6 h basal, 2 h glucose clamp). Endogenous lipolysis was blocked with acipimox and Intralipid was infused at varying rates (0, 3, 6 or 12 μL kg^?1 min^?1) to obtain four different levels of circulating FFAs. Endogenous growth hormone, insulin and glucagon secretion was blocked by somatostatin (300 μg h^?1) and replaced exogenously. 15N‐phenylalanine, 2H4‐tyrosine and 13C‐urea were infused continuously to assess protein turnover and ureagenesis. Results: We obtained four distinct levels of FFA concentrations ranging from 0.03 to 2.1 mmol L^?1 and 3‐hydroxybutyrate concentrations from 10 to 360 μmol L^?1. Whole‐body phenylalanine turnover and phenylalanine‐to‐tyrosine degradation decreased with increasing FFA levels as did insulin‐stimulated forearm fluxes of phenylalanine. Phenylalanine, tyrosine and urea concentrations also decreased progressively, whereas urea turnover was unperturbed. Conclusion: Circulating FFAs decrease amino acid concentrations and inhibit whole‐body phenylalanine fluxes and phenylalanine‐to‐tyrosine conversion. Our data cover FFA concentrations from 0 to 2 mmol L^?1 and indicate that FFAs exert their protein conserving effects in the upper physiological range (>1.5 mmol L^?1).     

Day-to-day variation in oxygen consumption at submaximal loads during ergometer cycling by adolescents.

The day-to-day variation in oxygen consumption (VO2) during ergometer cycling by 20 healthy adolescents, 10 females and 10 males, was measured using indirect calorimetry. The two sets of measurements were performed on two consecutive days. Great care was taken to minimize possible disturbing factors. Cycling started at 50 and 100 W for female and male adolescents, respectively. The load was increased at a rate of 5 W 30 s(-1). In order to reach steady state, the load was kept constant for 3.5 min twice during the cycling session, at 100 and 130 W for the females and at 130 and 160 W for the males. Cycling continued until exhaustion. The maximal loads were 196 W (mean) and 271 W (mean) for females and males, respectively. At the maximal loads the day-to-day variation (+/-2 SD) in oxygen consumption (VO2) was +/-330 ml min(-1) for females and 390 ml min(-1) for males. At the submaximal loads the day-to-day variation in heart rate (HR) was 9.3 beats min(-1) (+/-2 SD) (coefficient of variation, CV=3.4% at 130 W) for both sexes. The day-to-day variation in oxygen consumption (VO2) was +/-199 ml min(-1) (+/-2 SD) at the different submaximal loads and did not differ between female and male adolescents (CV=5.7% at 130 W). This natural day-to-day variation must be taken into consideration when using a submaximal ergometer cycling test for the evaluation of physical capacity in the two sexes.