A microbiome-directed therapeutic food for children recovering from severe acute malnutrition.

Severe acute malnutrition (SAM), defined anthropometrically as a weight-for-length z-score more than 3 standard deviations below the mean (WLZ<-3), affects 19 million children under 5-years-old worldwide. Complete anthropometric recovery after standard inventions is rare with children often left with moderate acute malnutrition (MAM; WLZ -2 to -3). Here we conduct a randomized controlled trial (RCT), involving 12-18-month-old Bangladeshi children from urban and rural sites, who after hospital-based treatment for SAM received a 3-month intervention with a microbiota-directed complementary food (MDCF-2) or a ready-to-use supplementary food (RUSF) as they transitioned to MAM. The rate of WLZ improvement was significantly greater with MDCF-2 than the more calorically-dense RUSF, as we observed in a previous RCT of Bangladeshi children with MAM without antecedent SAM. A correlated meta-analysis of aptamer-based measurements of 4,520 plasma proteins in this and the prior RCT revealed 215 proteins positively-associated with WLZ (prominently those involved in musculoskeletal and CNS development) and 44 negatively-associated proteins (related to immune activation), with a significant enrichment in levels of the positively WLZ-associated proteins in the MDCF-2 arm. Characterizing changes in 754 bacterial metagenome-assembled genomes in serially collected fecal samples disclosed the effects of acute rehabilitation for SAM on the microbiome, its transition as each child achieves a state of MAM, and how specific strains of Prevotella copri function at the intersection between MDCF-2 glycan metabolism and the rescue of growth faltering. These results provide a rationale for further testing the generalizability of the efficacy of MDCF and identify biomarkers for defining treatment responses.

Targeting complement C3a receptor resolves mitochondrial hyperfusion and subretinal microglial activation in progranulin-deficient frontotemporal dementia.

Mutations in progranulin ( GRN ) cause frontotemporal dementia ( GRN -FTD) due to deficiency of the pleiotropic protein progranulin. GRN -FTD exhibits diverse pathologies including lysosome dysfunction, lipofuscinosis, microgliosis, and neuroinflammation. Yet, how progranulin loss causes disease remains unresolved. Here, we report that non-invasive retinal imaging of GRN -FTD patients revealed deficits in photoreceptors and the retinal pigment epithelium (RPE) that correlate with cognitive decline. Likewise, Grn -/- mice exhibit early RPE dysfunction, microglial activation, and subsequent photoreceptor loss. Super-resolution live imaging and transcriptomic analyses identified RPE mitochondria as an early driver of retinal dysfunction. Loss of mitochondrial fission protein 1 (MTFP1) in Grn -/- RPE causes mitochondrial hyperfusion and bioenergetic defects, leading to NF-kB-mediated activation of complement C3a-C3a receptor signaling, which drives further mitochondrial hyperfusion and retinal inflammation. C3aR antagonism restores RPE mitochondrial integrity and limits subretinal microglial activation. Our study identifies a previously unrecognized mechanism by which progranulin modulates mitochondrial integrity and complement-mediated neuroinflammation.

A shared group of bacterial taxa in the duodenal microbiota of undernourished Pakistani children with environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z-score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A "core" group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans, Streptococcus australis, Granulicatella elegans, Granulicatella adiacens, and Abiotrophia defectiva. At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus, Gemella, and Granulicatella species, warrants further investigation.IMPORTANCEUndernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications.

Prevotella copri and microbiota members mediate the beneficial effects of a therapeutic food for malnutrition.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.

Bioactive glycans in a microbiome-directed food for children with malnutrition.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.

An evolution-based framework for describing human gut bacteria.

The human gut microbiome contains many bacterial strains of the same species ('strain-level variants'). Describing strains in a biologically meaningful way rather than purely taxonomically is an important goal but challenging due to the genetic complexity of strain-level variation. Here, we measured patterns of co-evolution across >7,000 strains spanning the bacterial tree-of-life. Using these patterns as a prior for studying hundreds of gut commensal strains that we isolated, sequenced, and metabolically profiled revealed widespread structure beneath the phylogenetic level of species. Defining strains by their co-evolutionary signatures enabled predicting their metabolic phenotypes and engineering consortia from strain genome content alone. Our findings demonstrate a biologically relevant organization to strain-level variation and motivate a new schema for describing bacterial strains based on their evolutionary history.

Prevotella copri-related effects of a therapeutic food for malnutrition.

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

Bioactive glycans in a microbiome-directed food for malnourished children.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Designing effective microbiome-directed therapeutic foods to repair these perturbations requires knowledge about how food components interact with the microbiome to alter its expressed functions. Here we use biospecimens from a randomized, controlled trial of a microbiome-directed complementary food prototype (MDCF-2) that produced superior rates of weight gain compared to a conventional ready-to-use supplementary food (RUSF) in 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM)4. We reconstructed 1000 bacterial genomes (metagenome-assembled genomes, MAGs) present in their fecal microbiomes, identified 75 whose abundances were positively associated with weight gain (change in weight-for-length Z score, WLZ), characterized gene expression changes in these MAGs as a function of treatment type and WLZ response, and used mass spectrometry to quantify carbohydrate structures in MDCF-2 and feces. The results reveal treatment-induced changes in expression of carbohydrate metabolic pathways in WLZ-associated MAGs. Comparing participants consuming MDCF-2 versus RUSF, and MDCF-2-treated children in the upper versus lower quartiles of WLZ responses revealed that two Prevotella copri MAGs positively associated with WLZ were principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilization of its component glycans. Moreover, the predicted specificities of carbohydrate active enzymes expressed by polysaccharide utilization loci (PULs) in these two MAGs correlate with the (i) in vitro growth of Bangladeshi P. copri strains, possessing differing degrees of PUL and overall genomic content similarity to these MAGs, cultured in defined medium containing different purified glycans representative of those in MDCF-2, and (ii) levels of carbohydrate structures identified in feces from clinical trial participants. In the accompanying paper5, we use a gnotobiotic mouse model colonized with age- and WLZ-associated bacterial taxa cultured from this study population, and fed diets resembling those consumed by study participants, to directly test the relationship between P. copri, MDCF-2 glycan metabolism, host ponderal growth responses, and intestinal gene expression and metabolism. The ability to identify bioactive glycan structures in MDCFs that are metabolized by growth-associated bacterial taxa will help guide recommendations about use of this MDCF for children with acute malnutrition representing different geographic locales and ages, as well as enable development of bioequivalent, or more efficacious, formulations composed of culturally acceptable and affordable ingredients.

Evaluating microbiome-directed fibre snacks in gnotobiotic mice and humans.

Changing food preferences brought about by westernization that have deleterious health effects1,2-combined with myriad forces that are contributing to increased food insecurity-are catalysing efforts to identify more nutritious and affordable foods3. Consumption of dietary fibre can help to prevent cardiovascular disease, type 2 diabetes and obesity4-6. A substantial number of reports have explored the effects of dietary fibre on the gut microbial community7-9. However, the microbiome is complex, dynamic and exhibits considerable intra- and interpersonal variation in its composition and functions. The large number of potential interactions between the components of the microbiome makes it challenging to define the mechanisms by which food ingredients affect community properties. Here we address the question of how foods containing different fibre preparations can be designed to alter functions associated with specific components of the microbiome. Because a marked increase in snack consumption is associated with westernization, we formulated snack prototypes using plant fibres from different sustainable sources that targeted distinct features of the gut microbiomes of individuals with obesity when transplanted into gnotobiotic mice. We used these snacks to supplement controlled diets that were consumed by adult individuals with obesity or who were overweight. Fibre-specific changes in their microbiomes were linked to changes in their plasma proteomes indicative of an altered physiological state.

A Microbiota-Directed Food Intervention for Undernourished Children.

BACKGROUND: More than 30 million children worldwide have moderate acute malnutrition. Current treatments have limited effectiveness, and much remains unknown about the pathogenesis of this condition. Children with moderate acute malnutrition have perturbed development of their gut microbiota. METHODS: In this study, we provided a microbiota-directed complementary food prototype (MDCF-2) or a ready-to-use supplementary food (RUSF) to 123 slum-dwelling Bangladeshi children with moderate acute malnutrition between the ages of 12 months and 18 months. The supplementation was given twice daily for 3 months, followed by 1 month of monitoring. We obtained weight-for-length, weight-for-age, and length-for-age z scores and mid-upper-arm circumference values at baseline and every 2 weeks during the intervention period and at 4 months. We compared the rate of change of these related phenotypes between baseline and 3 months and between baseline and 4 months. We also measured levels of 4977 proteins in plasma and 209 bacterial taxa in fecal samples. RESULTS: A total of 118 children (59 in each study group) completed the intervention. The rates of change in the weight-for-length and weight-for-age z scores are consistent with a benefit of MDCF-2 on growth over the course of the study, including the 1-month follow-up. Receipt of MDCF-2 was linked to the magnitude of change in levels of 70 plasma proteins and of 21 associated bacterial taxa that were positively correlated with the weight-for-length z score (P<0.001 for comparisons of both protein and bacterial taxa). These proteins included mediators of bone growth and neurodevelopment. CONCLUSIONS: These findings provide support for MDCF-2 as a dietary supplement for young children with moderate acute malnutrition and provide insight into mechanisms by which this targeted manipulation of microbiota components may be linked to growth. (Supported by the Bill and Melinda Gates Foundation and the National Institutes of Health; ClinicalTrials.gov number, NCT04015999.).

Duodenal Microbiota in Stunted Undernourished Children with Enteropathy.

BACKGROUND: Environmental enteric dysfunction (EED) is an enigmatic disorder of the small intestine that is postulated to play a role in childhood undernutrition, a pressing global health problem. Defining the incidence of this disorder, its pathophysiological features, and its contribution to impaired linear and ponderal growth has been hampered by the difficulty in directly sampling the small intestinal mucosa and microbial community (microbiota). METHODS: In this study, among 110 young children (mean age, 18 months) with linear growth stunting who were living in an urban slum in Dhaka, Bangladesh, and had not benefited from a nutritional intervention, we performed endoscopy in 80 children who had biopsy-confirmed EED and available plasma and duodenal samples. We quantified the levels of 4077 plasma proteins and 2619 proteins in duodenal biopsy samples obtained from these children. The levels of bacterial strains in microbiota recovered from duodenal aspirate from each child were determined with the use of culture-independent methods. In addition, we obtained 21 plasma samples and 27 fecal samples from age-matched healthy children living in the same area. Young germ-free mice that had been fed a Bangladeshi diet were colonized with bacterial strains cultured from the duodenal aspirates. RESULTS: Of the bacterial strains that were obtained from the children, the absolute levels of a shared group of 14 taxa (which are not typically classified as enteropathogens) were negatively correlated with linear growth (length-for-age z score, r = -0.49; P = 0.003) and positively correlated with duodenal proteins involved in immunoinflammatory responses. The representation of these 14 duodenal taxa in fecal microbiota was significantly different from that in samples obtained from healthy children (P<0.001 by permutational multivariate analysis of variance). Enteropathy of the small intestine developed in gnotobiotic mice that had been colonized with cultured duodenal strains obtained from children with EED. CONCLUSIONS: These results provide support for a causal relationship between growth stunting and components of the small intestinal microbiota and enteropathy and offer a rationale for developing therapies that target these microbial contributions to EED. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov number, NCT02812615.).

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.

Endoscopic submucosal dissection: experience in an Australian tertiary center.

BACKGROUND: Endoscopic submucosal dissection (ESD) is a technique for en bloc resection of superficial tumors of the gastrointestinal tract. In contrast to Japan and other Asian countries, few data are available in Western countries. The objective of the current study was to evaluate the experience of ESD in a single Australian tertiary center. METHODS: The patient features, outcomes and complications of ESD of 20 lesions in 18 patients at a single center between 2008 and 2012, were evaluated retrospectively. RESULTS: Twenty lesions, in 18 patients of median age 69.5 years, were resected with ESD. Ten patients had gastric lesions (of whom two had two synchronous lesions), four patients had rectal lesions, one had a colonic lesion, one had a duodenal lesion and two had esophageal lesions. The median (range) lesion size was 2.5 (0.5-6.5) cm. In the entire cohort, en bloc resection occurred in 80% cases and complete histological resection was achieved in 60% cases. Significant bleeding requiring repeat endoscopy and transfusion occurred in two cases and microscopic perforation occurred in 1 case. Surgery for unsuccessful ESD was pursued without complication in 6 cases. There were two recurrences during follow up of median 36 months, both of which occurred in cases of gastric ESD and one of which (carcinoid) occurred after surgery. CONCLUSIONS: ESD appears feasible in an Australian population. It should however be contemplated in carefully selected patients whilst there is refinement of pre-treatment diagnosis, the ESD technique and the management of its complications.

Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration.

The regenerative capacity of skeletal muscle declines with age. Previous studies suggest that this process can be reversed by exposure to young circulation; however, systemic age-specific factors responsible for this phenomenon are largely unknown. Here we report that oxytocin--a hormone best known for its role in lactation, parturition and social behaviours--is required for proper muscle tissue regeneration and homeostasis, and that plasma levels of oxytocin decline with age. Inhibition of oxytocin signalling in young animals reduces muscle regeneration, whereas systemic administration of oxytocin rapidly improves muscle regeneration by enhancing aged muscle stem cell activation/proliferation through activation of the MAPK/ERK signalling pathway. We further show that the genetic lack of oxytocin does not cause a developmental defect in muscle but instead leads to premature sarcopenia. Considering that oxytocin is an FDA-approved drug, this work reveals a potential novel and safe way to combat or prevent skeletal muscle ageing.

Longitudinal observation of viral load changes in untreated HBeAg negative chronic hepatitis B.

INTRODUCTION: An HBV DNA level of 2000 IU/ml has been used to differentiate HBeAg negative chronic hepatitis B from the inactive carrier state. We sought to examine the nature and frequency of fluctuations in viral load and ALT around this threshold. METHODS: A retrospective review of St Vincent's Hospital database was performed to identify patients who had been observed, untreated, with HBV DNA and ALT levels over a period of at least 18 months. RESULTS: 27 HBeAg negative patients with HBV DNA < 2000 IU/ ml at baseline (Group 1) and 20 HBeAg negative patients with HBV DNA > or = 2000 IU/ml (Group 2) were identified. Of group 1 patients, only 8/27 had persistently normal ALT and HBV DNA persistently <2000 IU/ml over a median followup of 24 months. 11/27 (41%) Group 1 patients showed fluctuations above 2000 IU/ml over a median of 24 months followup, most of which were transient and in the range <20,000 IU/ml. They were accompanied by persistently normal ALT in 5/11 (45%). 8 of 20 (40%) Group 2 patients had a drop of HBV DNA to <2000 IU/ml over followup. These had a significantly lower baseline HBV DNA (8610 v/s 208763, p = 0.03) than those that remained persistently >2000 IU/ml. CONCLUSIONS: Minor fluctuations in HBV DNA up to 20,000 IU/ ml, accompanied by persistently normal ALT occur frequently in HBeAg negative chronic hepatitis B.

DNA methyltransferase-3-dependent nonrandom template segregation in differentiating embryonic stem cells.

Asymmetry of cell fate is one fundamental property of stem cells, in which one daughter cell self-renews, whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms, such as plants, fungi, and mammals, has already been shown. However, before this current work, asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs), and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing, differentiating human and mouse ESCs. Moreover, we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3), indicating a molecular mechanism that regulates this phenomenon. Furthermore, our data support the hypothesis that retention of chromatids with the "old" template DNA preserves the epigenetic memory of cell fate, whereas localization of "new" DNA strands and de novo DNA methyltransferase to the lineage-destined daughter cell facilitates epigenetic adaptation to a new cell fate.

Using patient management as a surrogate for patient health outcomes in diagnostic test evaluation.

BACKGROUND: Before a new test is introduced in clinical practice, evidence is needed to demonstrate that its use will lead to improvements in patient health outcomes. Studies reporting test accuracy may not be sufficient, and clinical trials of tests that measure patient health outcomes are rarely feasible. Therefore, the consequences of testing on patient management are often investigated as an intermediate step in the pathway. There is a lack of guidance on the interpretation of this evidence, and patient management studies often neglect a discussion of the limitations of measuring patient management as a surrogate for health outcomes. METHODS: We discuss the rationale for measuring patient management, describe the common study designs and provide guidance about how this evidence should be reported. RESULTS: Interpretation of patient management studies relies on the condition that patient management is a valid surrogate for downstream patient benefits. This condition presupposes two critical assumptions: the test improves diagnostic accuracy; and the measured changes in patient management improve patient health outcomes. The validity of this evidence depends on the certainty around these critical assumptions and the ability of the study design to minimise bias. Three common designs are test RCTs that measure patient management as a primary endpoint, diagnostic before-after studies that compare planned patient management before and after testing, and accuracy studies that are extended to report on the actual treatment or further tests received following a positive and negative test result. CONCLUSIONS: Patient management can be measured as a surrogate outcome for test evaluation if its limitations are recognised. The potential consequences of a positive and negative test result on patient management should be pre-specified and the potential patient benefits of these management changes clearly stated. Randomised comparisons will provide higher quality evidence about differences in patient management using the new test than observational studies. Regardless of the study design used, the critical assumption that patient management is a valid surrogate for downstream patient benefits or harms must be discussed in these studies.