Pre-analytic decrease of phenylalanine in plasma of patients with phenylketonuria treated with pegvaliase.

Treatment of phenylketonuria (PKU) has evolved since the initial introduction of a phenylalanine (Phe) restricted diet. The most recent option for adults affected with PKU is treatment with an alternate enzyme, phenylalanine ammonia lyase (PAL), that metabolizes excess Phe. Proper management of all patients with PKU relies on accurate measurement of Phe levels in blood, to comply with guidance intended to minimize the neurological symptoms. Recently, our laboratory was notified of discrepant results for a patient with PKU who is treated with pegvaliase. Two specimens were collected at the same time but yielded unexpectedly different Phe concentrations. After exclusion of specimen mix-ups or analytical errors, we suspected that there was residual pegvaliase activity in the specimens continuing to degrade Phe after collection. To investigate this possibility, we performed spiking studies that showed the degradation of Phe over time at ambient temperatures. Sample preparation by protein crash appears to deactivate pegvaliase and prevents further Phe degradation. However, because pegvaliase deactivation would be required immediately following blood collection, appropriate mitigation measures must be implemented, including stringent pre-analytical requirements, alternate sample matrices such as dried blood spots, or point of care testing. Until then, health care professionals need to be cautious in their interpretation of Phe levels in their patients with PKU that are treated with pegvaliase.

National HIV and HCV Screening Rates for Hospitalized People who Use Drugs Are Suboptimal and Heterogeneous Across 11 US Hospitals.

Background: To end the HIV and hepatitis C virus (HCV) epidemics, people who use drugs (PWUD) need more opportunities for testing. While inpatient hospitalizations are an essential opportunity to test people who use drugs (PWUD) for HIV and HCV, there is limited research on rates of inpatient testing for HIV and HCV among PWUD. Methods: Eleven hospital sites were included in the study. Each site created a cohort of inpatient encounters associated with injection drug use. From these cohorts, we collected data on HCV and HIV testing rates and HIV testing consent policies from 65 276 PWUD hospitalizations. Results: Hospitals had average screening rates of 40% for HIV and 32% for HCV, with widespread heterogeneity in screening rates across facilities. State consent laws and opt-out testing policies were not associated with statistically significant differences in HIV screening rates. On average, hospitals that reflexed HCV viral load testing on HCV antibody testing did not have statistically significant differences in HCV viral load testing rates. We found suboptimal testing rates during inpatient encounters for PWUD. As treatment (HIV) and cure (HCV) are necessary to end these epidemics, we need to prioritize understanding and overcoming barriers to testing.

SLC6A8 creatine transporter deficiency can be detected by plasma creatine and creatinine concentrations.

Creatine transporter deficiency has been described with normal or uninformative levels of creatine and creatinine in plasma, while urine has been the preferred specimen type for biochemical diagnosis. We report a cohort of untreated patients with creatine transporter deficiency and abnormal plasma creatine panel results, characterized mainly by markedly decreased plasma creatinine. We conclude that plasma should be considered a viable specimen type for the biochemical diagnosis of this disorder, and abnormal results should be followed up with further confirmatory testing.

A new test method for biochemical analysis of plasmalogens in dried blood spots and erythrocytes from patients with peroxisomal disorders.

Measurement of plasmalogens is useful for the biochemical diagnosis of rhizomelic chondrodysplasia punctata (RCDP) and is also informative for Zellweger spectrum disorders (ZSD). We have developed a test method for the simultaneous quantitation of C16:0, C18:0, and C018:1 plasmalogen (PG) species and their corresponding fatty acids (FAs) in dried blood spots (DBS) and erythrocytes (RBC) by using capillary gas chromatography-mass spectrometry. Normal reference ranges for measured markers and 10 calculated ratios were established by the analysis of 720 and 473 unaffected DBS and RBC samples, respectively. Determination of preliminary disease ranges was made by using 45 samples from 43 unique patients: RCDP type 1 (DBS: 1 mild, 17 severe; RBC: 1 mild, 6 severe), RCDP type 2 (DBS: 2 mild, 1 severe; RBC: 2 severe), RCDP type 3 (DBS: 1 severe), RCDP type 4 (RBC: 2 severe), and ZSD (DBS: 3 severe; RBC: 2 mild, 7 severe). Postanalytical interpretive tools in Collaborative Laboratory Integrated Reports (CLIR) were used to generate an integrated score and a likelihood of disease. In conjunction with a review of clinical phenotype, phytanic acid, and very long-chain FA test results, the CLIR analysis allowed for differentiation between RCDP and ZSD. Data will continue to be gathered to improve CLIR analysis as more samples from affected patients with variable disease severity are analyzed. The addition of DBS analysis of PGs may allow for at-home specimen collection and second-tier testing for newborn screening programs.

Transitioning from Medicaid to private health insurance: informing public and private sector outreach.

This study explored the lived experiences of transitioning from Medicaid to private health insurance upon college graduation. Fifteen recent graduates of an urban, commuter, public college in the Mid-Atlantic were interviewed via Zoom® to understand what they regard as crucial aspects of the transition experience, especially during the COVID pandemic. The subjects all identified as being members of a minority racial or ethnic group, the average age was 33 years (SD = 10.96), and all but one interview subject majored in the health sciences. Every recent graduate reported experiencing difficulty in the transition. Subjects felt unprepared for the transition, alone, and without support. "Copays" was the most common response to questions, frequently said with arms in the air for emphasis, as if the word "copay" summarized all of the lack of preparation, difficulty, and expense of the healthcare system after previously receiving Medicaid (i.e., free healthcare). The findings inform how the private sector should on-board new college graduates. There is a need for Medicaid case officers to better prepare clients for the transition and for human resources personnel in the private sector to sufficiently explain how private health insurance works.

Progressive brain atrophy and severe neurodevelopmental phenotype in siblings with biallelic COASY variants.

Biallelic pathogenic variants in the COASY gene have been associated with two distinct disease phenotypes, that is, COASY-protein associated neurodegeneration (CoPAN) and pontocerebellar hypoplasia type 12 (PCH 12). We present two siblings that independently presented with significant hypotonia and respiratory insufficiency at birth. Comprehensive genetic testing revealed homozygous variants within COASY, however, the progressive clinical and neuroradiologic findings described here are unique and have not been described previously. Magnetic resonance imaging showed progressive diffuse parenchymal loss throughout the bilateral cerebral hemispheres and atrophy of the basal ganglia and brainstem. As such, this article brings forth two additional cases of COASY-related disorder with abnormal newborn screening acylcarnitine profiles resembling carnitine palmitoyl transferase 1a (CPT1a) deficiency in two siblings who presented at birth with contractures, marked hypotonia and absent respiratory drive.

The Association of Preoperative Magnetic Resonance Imaging (MRI) With Surgical Management in Patients With Early-Stage Breast Cancer.

INTRODUCTION: The rate of mastectomy in lumpectomy-eligible patients with unilateral breast cancer is increasing. We sought to investigate the association between magnetic resonance imaging (MRI) and surgical management of patients with early-stage breast cancer by comparing the rate of mastectomy as first surgery in patients with and without preoperative MRI. METHODS: A bi-institutional retrospective study included patients diagnosed between 2016 and 2020. Lumpectomy-eligible patients with in situ and invasive cancer were included. Those receiving preoperative therapy, MRI before diagnosis, or with known bilateral cancer were excluded. The risk factors for bilateral and multicentric disease were accounted for. Fisher's exact and chi-square tests compared categorical variables, Wilcoxon two-sample test analyzed continuous variables, and multivariate analyses were performed with Poisson regression. RESULTS: Four hundred twenty-eight participants met inclusion criteria. Patients who received MRI were younger (58 versus 67 y; P < 0.001) and had denser breasts (group 3 or 4; 61% versus 25%; P < 0.001). Mastectomy rate was twice as high in patients undergoing MRI (32% versus 15%, rate ratio 2.16; P < 0.001), which remained significant in multivariate analysis (rate ratio 2.0; P < 0.001). Contralateral mastectomy (12% versus 4%; P = 0.466) and reexcision (13% versus 12%; P = 0.519) rates were similar. Time to surgery was greater in those receiving MRI alone and MRI biopsy (34 [no MRI] versus 45 [MRI] versus 62 [MRI biopsy]; P < 0.001 for both). CONCLUSIONS: MRI receipt is associated with a doubled rate of mastectomy in lumpectomy-eligible patients. Future work is needed to standardize patient selection for MRI to those with the highest likelihood of having additional undiagnosed disease.

Clinical, biochemical and genetic characteristics of MOGS-CDG: a rare congenital disorder of glycosylation.

PURPOSE: To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. METHODS: Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. RESULTS: Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. CONCLUSION: The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.

Locoregional delivery of CAR-T cells in the clinic.

Cellular therapies utilizing T cells expressing chimeric antigen receptors (CARs) have garnered significant interest due to their clinical success in hematological malignancies. Unfortunately, this success has not been replicated in solid tumors, with only a small fraction of patients achieving complete responses. A number of obstacles to effective CAR-T cell therapy in solid tumors have been identified including tumor antigen heterogeneity, poor T cell fitness and persistence, inefficient trafficking and inability to penetrate into the tumor, immune-related adverse events due to on-target/off-tumor toxicity, and the immunosuppressive tumor microenvironment. Many preclinical studies have focused on improvements to CAR design to try to overcome some of these hurdles. However, a growing body of work has also focused on the use of local and/or regional delivery of CAR-T cells as a means to overcome poor T cell trafficking and inefficient T cell penetration into tumors. Most trials that incorporate locoregional delivery of CAR-T cells have targeted tumors of the central nervous system - repurposing an Ommaya/Rickham reservoir for repeated delivery of cells directly to the tumor cavity or ventricles. Hepatic artery infusion is another technique used for locoregional delivery to hepatic tumors. Locoregional delivery theoretically permits increased numbers of CAR-T cells within the tumor while reducing the risk of immune-related systemic toxicity. Studies to date have been almost exclusively phase I. The growing body of evidence indicates that locoregional delivery of CAR-T cells is both safe and feasible. This review focuses specifically on the use of locoregional delivery of CAR-T cells in clinical trials.

Intolerant baboons avoid observer proximity, creating biased inter-individual association patterns.

Social network analysis is an increasingly popular tool for behavioural ecologists exploring the social organisation of animal populations. Such analyses require data on inter-individual association patterns, which in wild populations are often collected using direct observations of habituated animals. This assumes observers have no influence on animal behaviour; however, our previous work showed that individuals in a habituated group of chacma baboons (Papio ursinus griseipes) displayed consistent and individually distinct responses to observer approaches. We explored the implications of our previous findings by measuring the inter-individual association patterns of the same group of chacma baboons at different observer distances. We found a strong positive association between individual tolerance levels (towards observers) and how often an animal appeared as a neighbour to focal animals when observers were nearer, and a neutral relationship between the same variables when the observer was further away. Additionally, association matrices constructed from different observation distances were not comparable within any proximity buffer, and neither were the individual network metrics generated from these matrices. This appears to be the first empirical evidence that observer presence and behaviour can influence the association patterns of habituated animals and thus have potentially significant impacts on measured social networks.

Identifying Policy Best-Practices to Support the Contribution of Aquatic Foods to Food and Nutrition Security.

The relationship between aquatic foods and food nutrition and security is increasingly recognised in policy and practice, yet many governance instruments do not acknowledge or support this important connection. The most effective policy approaches to support the link between these sectors, or 'best practices' are currently unknown. We reviewed relevant governance instruments from multiple countries to identify how these instruments linked fisheries, aquaculture and food security and nutrition, including the policy framing and evidence of political commitment. Of the documents connecting the sectors (65%), the majority did so in the context of developing the fisheries/aquaculture sector to increase aquatic food availability and/or access (51%), followed by developing the fisheries/aquaculture sector as a livelihoods approach to indirectly improve food security (33%), for example, through income generation. Sectoral links established in the context of nutrition-sensitive approaches to fisheries and aquaculture were less common (5%). Almost one third (29%) of instruments supported the connection between aquatic foods and food security and nutrition across three or more different contexts relevant to food security or food systems, while 12% indicated a very high level of commitment. We recommend some key attributes for future policy development to help build coherence between sectors and to help frame coherent food system-based policies.

Biochemical Diagnosis of Acute Hepatic Porphyria: Updated Expert Recommendations for Primary Care Physicians.

Acute hepatic porphyria (AHP) is a group of rare, metabolic diseases where patients can experience acute neurovisceral attacks, chronic symptoms, and long-term complications. Diagnostic biochemical testing is widely available and effective, but a substantial time from symptom onset to diagnosis often delays treatment and increases morbidity. A panel of laboratory scientists and clinical AHP specialists collaborated to produce recommendations on how to enhance biochemical diagnosis of AHP in the USA. AHP should be considered in the differential diagnosis of unexplained abdominal pain, the most common symptom, soon after excluding common causes. Measurement of porphobilinogen (PBG) and porphyrins in a random urine sample, with results normalized to creatinine, is recommended as an effective and cost-efficient initial test for AHP. Delta-aminolevulinic acid testing may be included but is not essential. The optimal time to collect a urine sample is during an attack. Substantial PBG elevation confirms an AHP diagnosis and allows for prompt treatment initiation. Additional testing can determine AHP subtype and identify at-risk family members. Increased awareness of AHP and correct diagnostic methods will reduce diagnostic delay and improve patient outcomes.

Incorporation of Second-Tier Biomarker Testing Improves the Specificity of Newborn Screening for Mucopolysaccharidosis Type I.

Enzyme-based newborn screening for Mucopolysaccharidosis type I (MPS I) has a high false-positive rate due to the prevalence of pseudodeficiency alleles, often resulting in unnecessary and costly follow up. The glycosaminoglycans (GAGs), dermatan sulfate (DS) and heparan sulfate (HS) are both substrates for α-l-iduronidase (IDUA). These GAGs are elevated in patients with MPS I and have been shown to be promising biomarkers for both primary and second-tier testing. Since February 2016, we have measured DS and HS in 1213 specimens submitted on infants at risk for MPS I based on newborn screening. Molecular correlation was available for 157 of the tested cases. Samples from infants with MPS I confirmed by IDUA molecular analysis all had significantly elevated levels of DS and HS compared to those with confirmed pseudodeficiency and/or heterozygosity. Analysis of our testing population and correlation with molecular results identified few discrepant outcomes and uncovered no evidence of false-negative cases. We have demonstrated that blood spot GAGs analysis accurately discriminates between patients with confirmed MPS I and false-positive cases due to pseudodeficiency or heterozygosity and increases the specificity of newborn screening for MPS I.

The critical role of psychosine in screening, diagnosis, and monitoring of Krabbe disease.

PURPOSE: Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations. METHODS: PSY was extracted from dried blood spots or erythrocytes with methanol containing d5-PSY as internal standard, and measured by liquid chromatography-tandem mass spectrometry. RESULTS: Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT). CONCLUSION: This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.

Multiplex testing for the screening of lysosomal storage disease in urine: Sulfatides and glycosaminoglycan profiles in 40 cases of sulfatiduria.

PURPOSE: To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine. METHODS: Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results. RESULTS: Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders. CONCLUSIONS: Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans.

3'-Modification stabilizes mRNA and increases translation in cells.

Successful implementation of mRNA gene therapy is facing many hurdles, for example poor expression levels of the exogenously delivered mRNA transcripts. Herein we describe the synthesis of various 3'-modified RNA oligonucleotides, and we show that 3'-modification drastically stabilizes these oligonucleotides in cell extracts. Modification of the 3'-terminus of gaussia luciferase mRNA results in 3-fold increased and extended (>48 h) translation of the mRNA. Our findings suggest 3'-modification of RNA-transcripts as a valid approach to increase expression levels for application in mRNA gene therapy.