Preeclampsia remains a leading cause of maternal and perinatal morbidity and mortality. Accurate prediction of preeclampsia risk would enable more effective, risk-based prenatal care pathways. Current risk assessment algorithms depend on clinical risk factors largely unavailable for first-time pregnant women. Delivering accurate preeclampsia risk assessment to this cohort of women, therefore requires for novel biomarkers. Here, we evaluated the relevance of metabolite biomarker candidates for their selection into a prototype rapid, quantitative Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based clinical screening assay. First, a library of targeted LC-MS/MS assays for metabolite biomarker candidates was developed, using a medium-throughput translational metabolomics workflow, to verify biomarker potential in the Screening-for-Pregnancy-Endpoints  study. A variable pre-selection step was followed by the development of multivariable prediction models for pre-defined clinical use cases, i.e., prediction of preterm preeclampsia risk and of any preeclampsia risk. Within a large set of metabolite biomarker candidates, we confirmed the potential of dilinoleoyl-glycerol and heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine to effectively complement Placental Growth Factor, an established preeclampsia biomarker, for the prediction of preeclampsia risk in first-time pregnancies without overt risk factors. These metabolites will be considered for integration in a prototype rapid, quantitative LC-MS/MS assay, and subsequent validation in an independent cohort. Preeclampsia remains a leading cause of maternal death throughout the world and is responsible for considerable neonatal morbidity and mortality . ManagemThe National Institute for Health and Clinical Excellence (NICE) in England and the American College of Obstetricians and Gynecologists (ACOG) in the United States recommend the use of maternal demographic, obstetrical history and overt medical risk factors in simple dichotomous classifications to determine low or high risk status for the development of preeclampsia , 6. HoweThe need for novel biomarkers to improve preeclampsia prediction in nulliparous women without apparent risk factor is further emphasized by the fact that nulliparity is associated with increased risk (RR = 2.1 (1.9 to 2.4)) and accounts for the largest single population attributable fraction (32.3%) for preeclampsia .To date, the most studied biomarker in preeclampsia is Placental Growth Factor (PlGF), the levels of which tend to drop in a sub-group of women destined to develop preeclampsia , and whiin vitro diagnostic workflows already operated in clinical laboratories.The aim of this work was to inform the development of a novel biomarker-assisted test for the early and accurate prediction of preeclampsia risk in low-risk nulliparous women. With preeclampsia recognised as being a syndromic condition, accurate prediction of preeclampsia will require for several biomarkers to account for different preeclampsia subtypes and/or patient risk profiles –21. BecaWe previously reported a metabolomic biomarker discovery study for early pregnancy prediction of later preeclampsia . BuildinFirstly we developed a translational research workflow based on LC-QqQ-MS based metabolite quantification; only candidate biomarkers amenable to this workflow were considered for further analysis in a case-control study. Biomarkers with univariable prediction merits were pre-selected for the development of risk predictor models. Models were evaluated in function of two pre-defined clinical use scenarios: (1) identify women at risk of developing preterm preeclampsia (primary objective), and (2) identify women at risk of developing preeclampsia at any stage of the pregnancy (secondary objective). We found that combining dilinoleoyl-glycerol (DLG) with PlGF effectively predicted increased preterm preeclampsia risk at ca. 15 weeks of gestation. The further addition of heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (1-HGP) expanded the capacity to also identify pregnant women at decreased risk of developing any form of preeclampsia. PlGF, DLG and 1-HGP were therefore proposed for progression to the final development stage of a risk prediction test for (preterm) preeclampsia and subsequent validation in the IMproved Pregnancy Outcomes by Early Detection (IMPROvED) study .Previously reported metabolomic biomarkers discovered in plasma samples from participants of the New Zealand and Australian Cohort of the Screening for Pregnancy Endpoints (SCOPE) study were considered . A curatAll samples were obtained from participants in the European centres of the SCOPE study  between November 2004 and August 2008. Blood samples were prospectively collected from low-risk nulliparous women between 14 weeks and zero days and 16 weeks and six days gestation (or 15±1 week). The diagnosis of preeclampsia was made at any stage during pregnancy after recruitment until delivery, or in the first 2 weeks postpartum.Preeclampsia was defined according to the International Society for the study of Hypertension in Pregnancy (ISSHP)  S2 FileS2 File. i) uncertainty of last menstrual period (LMP) and unwillingness to have an ultrasound scan at ≤ 20 weeks’ gestation; ii) known major fetal anomaly or abnormal karyotype; iii) underlying medical conditions , previous knife cone biopsy, ≥3 miscarriages or ≥3 terminations, current ruptured membranes, major uterine anomaly, cervical suture; and iv) treatment with: long term steroids, low-dose aspirin, calcium (>1 g/24h), eicosopentaenoic acid, vitamin C ≥1000 mg, vitamin E ≥ 400 IU, or low molecular weight heparin.The inclusion criteria were nulliparity, singleton pregnancy, gestational age between 14 weeks and zero days and 16 weeks and six days, and informed consent to participate. The exclusion criteria applied included: Ethics approval was gained from local ethics committees of each participating centre  05/02/08) and written informed consent was obtained from all participants. Collection of data and biological samples complied with standardised procedures in all participating centres and was conducted in accordance with the principles of the Declaration of Helsinki.Biobanked EDTA plasma samples from all participants with a confirmed pregnancy outcome of the European branch of SCOPE were available. Within the n = 2364 cohort of pregnant women, 97 developed preeclampsia and 2266 did not develop preeclampsia. EDTA plasma samples from the 97 preeclampsia case patients and 335 randomly selected non-preeclampsia control patients were considered for analysis in the case-control study. The demographic and clinical characteristics of the control population selected for the study were compared pairwise and the lack of significant bias was verified .rule-in classification to identify women at high risk of developing preterm preeclampsia  ≥0.05); (2a) a rule-in classification identifying women at high risk of developing preeclampsia at any stage of the pregnancy ; and (2b) a rule-out classification identifying women at low risk of developing preeclampsia at any stage of the pregnancy  ≥0.99). Given that the target pregnancy population constitutes healthy first-time pregnant women, the comparator for the different classifications was defined as prediction models constituting any of the following established predictors: PlGF, body mass index (BMI) and Mean Arterial Pressure (MAP): the second set of blood pressure measurements was used to calculate MAP, as reported earlier [In consultation with a panel of clinicians, two relevant clinical use scenarios were established for stratifying nulliparous pregnant women without overt risk factors to their preeclampsia risk. These translated to three performance criteria for eventual predictor models relevant to the intended use population: (1) a  earlier .Chemicals and reagents used were of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) grade or higher . Metabolite reference substances and stable isotope-labelled standards were purchased from Fluka , Fischer Scientific , IsoSciences , Sigma-Aldrich , Avanti Lipids , QMX Laboratories , LGC , Alfa Chemistry , Generon , Larodan  and R&D Systems .For sample preparation, a liquid handling robot, equipped with a 96 LT disposable Tip Head, an orbital shaker station and a Peltier Thermal Station, was used .Targeted metabolite LC-MS/MS analysis was performed using a 1260 Infinity LC system  coupled to an Agilent Triple Quadrupole 6460 mass spectrometer (QqQ-MS) equipped with a JetStream Electrospray Ionisation (ESI) source .An analytical workflow which complied with pre-defined requirements , calibrators and Quality Control samples (QCs) were re-configured in analytical batches of 96 samples. The positions of Calibrators (n = 8), Analytical QCs (2*3), and study pool QCs (n = 9) were kept identical across batches. Fifteen percent of clinical samples were selected for duplicate analysis. The samples, inclusive the duplicate samples, were randomised for the analytical process using stratified randomisation over clinical centre, ethnicity, and preeclampsia. The absence of potential sources of experimentally induced bias was confirmed by computing pairwise dependencies between the randomisation outputs  and clinical parameters; no significant associations were found. Further information on the study and sample preparation is given in All batches were first analysed by RPLC-MS/MS and then by HILIC-MS/MS. Following mass spectrometric analysis, the mass spectrometric signals of all measured quantifier and qualifier transitions of the 54 assay-able metabolites and 38 SIL-IS were quantified using a pre-defined quantification method compiled in Masshunter Quant Software vB.07.00  across all the samples analysed in the study. All data were then reviewed by two independent analysts and any erroneous signal integration calls made by the software curated; all manual curation was recorded for data integrity purposes.PlGF was analysed earlier as part of a large-scale assessment of putative protein biomarkers within the SCOPE study using a Luminex Sandwich assay . The latAll laboratory personnel were blinded to sample status (case or control) at all stages of the study. Structured review of the calibrator signal data and signal data across all sample types was performed to confirm metabolite quantifier transition selection, absence of interference in the SIL-IS transitions, and final metabolite quantification metric. Next, pairwise dependencies between metabolite quantification metrics and recorded experimental variables were computed using the appropriate statistical tests; no aberrant correlations were found other than some minor inter-day batch effects in some of the quantifications. For the metabolite quantifications exhibiting batch-to-batch differences, the relative concentrations were scaled per batch using the ratio of the median concentration for the given batch over the overall median concentration .Data missingness and imprecision criteria were assessed: data missingness for a given metabolite quantification should be < 20% across all clinical samples, with the exception of cotinine, a reporter metabolite for smoking status. Two imprecision metrics, expressed as coefficient of variance (CV), were calculated: 1) for the replicate study-QC pool samples across all batches and 2) for the 15% technical duplicates . Metabolites were selected when %CV ≤ 25% for both metrics. Summary of the assay selection for biomarker analyses based on quality assurance evaluation is provided in Statistical analyses were performed using the statistical software R version 3.5 .0, of 1 and 2 x 104 permutations [In a first phase, variables were evaluated based on their stand-alone predictive performance for preterm preeclampsia (<37 weeks of gestation), term preeclampsia (> = 37 weeks of gestation) and preeclampsia. Variables considered were: all analytes as well as the established risk factors for preeclampsia, blood pressure (MAP) and BMI. The discriminative performance of each individual variable was quantified using the area under the receiver operating curve (AUROC) . Variablutations .The concentrations and relative concentrations of analytes were log-transformed before any modelling. Analysis was performed on complete cases.First, multivariable models were developed for the outcome ‘any preeclampsia’ using partial least squares–discriminant analysis (PLS-DA) . ComparaAll models were evaluated for the clinical use cases defined. For all models, cut-off values for concentrations and scores were selected based on the rule-in PPV and rule-out NPV criteria. PPV, NPV, sensitivity and specificity, and their respective 95% confidence intervals were estimated at the given cut-offs using bootstrapping. For each model, the superiority of the rule-in sensitivity and rule-out superiority as compared to reference models was computed by estimating a difference in sensitivity or specificity using bootstrapping and paired T test . The reference models were the single marker PlGF, the linear model PlGF+DLG and the recursive partitioning models PlGF||DLG and PlGF||MAP||BMI respectively.A nested case-control study (n = 432) was performed within the 2364 women recruited into the European SCOPE cohort, for whom pregnancy outcomes were known. Twenty-three women developed preterm preeclampsia (1%) and seventy-four (4%) developed term preeclampsia. Background characteristics and outcomes of pregnancy in women who did and did not develop preeclampsia are shown in Initially, 58 previously identified putative metabolite biomarkers were selected as inputs for this translational research; metabolites without ready-accessible reference materials available were excluded, except for sphinganine-1-phosphate for which assay parameters could be inferred from sphingosine-1-phosphate, leaving fifty-two metabolites for assay development. An additional set of 20 putative metabolite biomarkers from other sources , deemed From the fifty-four (54) metabolites analysed in the case-control study, forty-two complied with the pre-set quality assurance criteria. Two exceptions were allowed, 25-Hydroxyvitamin D3 and Eicosapentaenoic acid, which missed the criterion in one of the two types of QC samples considered. One assay was removed due to being non-specific: the signal constituted contributions of 2-methylglutaric acid, glutamine and an unknown metabolite. Also, one opportunistic marker was assessed: there were no detectable levels for 1,2-Dioctanoyl-sn-glycero-3-phosphocholine, yet the same assay delivered a consistent signal at a slightly different retention time. Comparison with reference materials confirmed this compound to be 1-heptadecanoyl-glycero-3-phosphocholine, the latter was therefore analysed. This resulted in data for forty-three metabolites being progressed to biomarker evaluation .Biomarkers and biometric variables selected based on their stand-alone prediction performance of preterm, term or any preeclampsia are shown in Model development was targeted to answer two pre-set outcome based clinical use scenarios (preterm preeclampsia and any preeclampsia), and three associated minimal performance requirements, expressed as Positive and Negative Predictive Value thresholds . The multivariable models developed within this framework are shown in With regards to predicting increased risk for any type of preeclampsia at a pre-set PPV cut-off of ≥ 0.15 (secondary objective), comprehensive analysis of the generated prediction models revealed that no classifier compliant with the PPV≥0.15 requirement could be identified.In models developed to meet the “rule-out” performance criteria , specifiIn this study we re-evaluated the relevance of a set of putative metabolite biomarkers to further inform the development of a test to predict preeclampsia in nulliparous women without apparent risk factors. Upon application of both technical assay-ability and data quality assurance criteria, forty-three metabolites, PlGF and the biometric variables MAP and BMI, well-known to associate with preeclampsia, were retained as predictors for consideration.In line with the findings that placental pathology is more common in preterm preeclampsia , we adopThis led to an initial selection of twelve predictors, comprising the clinical risk factors relevant in the low-risk population studied , PlGF and nine metabolites from the original set of forty-three quantified metabolites. For all predictors but BMI, the single variables showed discriminative performance which aligned with the prediction of either preterm preeclampsia or term preeclampsia. This observation supports the assumption that in this study population the two disease subtypes considered do indeed align well with distinct underlying physiological pathways. Interestingly, we note that in this study population, MAP is only a stand-alone predictor for term preeclampsia and not for preterm preeclampsia, whereas typically MAP is reported to be a strong stand-alone predictor for preterm preeclampsia . With hirule-in classification to identify women at high risk of developing preterm preeclampsia. The secondary clinical use scenario was delivering the same preeclampsia risk information to nulliparous pregnant women without overt risk factors, as the risk information available to parous women based on their obstetrics history [Consultation with a panel of clinicians  established two relevant clinical use scenarios for stratifying nulliparous pregnant women without overt risk factors to their preeclampsia risk. The primary clinical use scenario was a  history .In risk stratification, the clinical usefulness of a test is best expressed in terms of positive and negative predictive values . Hence, Considering these “target” inputs led to the establishment of a set of hierarchical goals for the development of predictive models for preeclampsia in nulliparous women without apparent risk factors. The primary goal was defined as the identification of pregnant women at increased risk for preterm preeclampsia with a post-test risk ≥ 0.05 (or PPV ≥ 0.05). Secondary to this, the establishment of generic risk for developing preeclampsia at any stage of pregnancy. For the latter, both the identification of women at high risk, defined as a post-test risk ≥ 15% (PPV ≥ 0.15), and the identification of women at low risk, defined as a post-test risk ≤ 1% (NPV ≥ 0.99), were considered of interest to clinical practice.Among all preeclampsia biomarkers studied, PlGF is the best understood. Low PlGF levels throughout pregnancy are associated with a disease phenotype which has placental insufficiency as a key characteristic , 41–44. Given the multi-factorial nature of preeclampsia, a Bayesian modelling approach may deliver more accurate predictive models than PLS-DA , 45. HowWe indeed found that recursive partitioning delivered better prediction than PLS-DA models. By establishing PlGF as the first independent predictor, the recursive partitioning modelling allowed us to align the model development with the pre-defined classification objectives. We found that complementing PlGF with a single metabolite biomarker (DLG) increased the sensitivity of the test from 48% (at PPV = 0.05) to 74% (at PPV = 0.06) for predicting preterm preeclampsia risk in nulliparous women without overt risk factors. Preliminary results show combining 1-HGP with DLG in the second partition may further improve the prediction of preterm preeclampsia, increasing the sensitivity to 78%.Developing recursive partitioning models with three splits suggested that 1-HGP may also be a third independent predictor. Adding 1-HGP enabled identification of significant fraction of women at low risk of developing preeclampsia (specificity = 36%) in accordance with the pre-set rule-out target of NPV = 0.99.et al. In this model, pregnant women at risk of developing preeclampsia may experience an uncomplicated pregnancy, as their pregnancy  concluded prior to their preeclampsia risk coming to expression [In this data set we were unable to establish a prediction model which also complied with the other secondary goal: the identification of a sizeable group of women with a post-test risk ≥ 15% of developing any type of preeclampsia in their pregnancy. This may be explained in the context of the competing risk model concept as established for preeclampsia by Wright pression .It is however noted that the list of putative biomarkers considered here was primarily informed by our earlier work  and fromA review of the literature reveals that DLG and 1-HGP may map onto different but complementary pathways relative to PlGF. Genetic studies have provided new insights into the mechanisms that link intracellular diacylglycerol concentrations to insulin resistance , 52, whiBased on the data generated in this study, DLG and 1-HGP are selected as the key markers for the development of a dedicated, rapid, quantitative LC-MS/MS assay prototype.In this study, we adopted an end-user focused translational research methodology for the prioritization of putative metabolite biomarkers for further development into a clinical test for preeclampsia risk prediction. Three key determinants were considered in the selection process: compatibility with LC-MS/MS technology already available in clinical laboratories, ability to improve the prediction of the established preeclampsia biomarker PlGF, and ability to meet patient stratification requirements as identified by clinical practitioners.Using this translational research framework to re-evaluate fifty-eight metabolite biomarker candidates, we were able to prioritise two metabolite biomarkers from an initial pre-selection of nine metabolites, for development of a clinical assay. We found that the metabolites DLG and 1-HGP effectively complemented PlGF in a prediction model for (preterm) preeclampsia in nulliparous women without over risk factors. A review of the literature supports that PlGF, DLG and 1-HGP may represent separate pathways for preeclampsia risk in the population studied.A strength of this study lies in the well-defined pregnancy risk profile within the SCOPE study, allowing us to focus this translation biomarker research to the pregnancy population most in need for biomarker-assisted risk prediction. In addition, a rational approach considering both technical usability and clinical utility criteria for refining the selection of previously identified putative biomarkers of interest for further test development, was adopted to increase the chance of successfully porting novel biomarkers into clinical practice.A limitation of this work lies within the ethnic make-up of the SCOPE-Europe study; most of the women were of white ethnicity. In line with the hypothesis that different pregnancy populations may constitute different patient risk profiles, there may be a need for additional biomarkers, representing additional preeclampsia risk pathways, to improve prediction for other ethnicities. Another limitation of this study is the gestational age considered; risk prediction involved samples taken early in the second trimester of pregnancy (15 +/- 1 week of gestation), whereas there is trend to consolidate prenatal risk screening at the end of the first trimester. However, we note that none of the verified metabolite biomarkers show any dependency on gestational age of sampling .The next steps in our test development work will involve developing a multiplex clinical assay for the selected markers, as well as further assessment of their generalisability to complement PlGF for the prediction in preeclampsia in other pregnancy populations. In a first instance, the markers will be assessed in IMPROvED; a pan-European multi-centre phase 2A clinical study which recruited ca 4000 low risk primiparous women for the purposes of assessing and refining innovative biomarker-assisted prototype predictive tests .S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Indirect estimation of reference intervals (RIs) is straightforward and inexpensive procedure for determination of intra-laboratory RIs. We applied the indirect approach to assess RIs for haematological parameters in capillary blood of pre-school children, using results stored in our laboratory database.We extracted data from laboratory information system, for the results obtained by automatic haematology analyser in capillary blood of 154 boys and 146 girls during pre-school medical examination. Data distribution was tested, and logarithmic transformation was applied if needed. Reference intervals were calculated by the nonparametric percentile method.12/L), haemoglobin (114-146 g/L), MCH (25.0-29.4 pg), MCHC (321-368 g/L), RDW-SD (36.1-43.5 fL), WBC count (4.5-12.3 x109/L), neutrophils count (1.7-6.9 x109/L) and percentage (29.0-69.0%), lymphocytes count (1.6-4.4 x109/L) and percentage (21.9-60.7%), PLT (165-459 x109/L), MPV (8.1-11.4 fL) and PDW (9.2-14.4%). Gender specific RIs were calculated for monocytes count : 0.2-1.6 x109/L; female (F): 0.1-1.4 x109/L) and percentage , haematocrit , MCV  and RDW , due to observed gender differences in these parameters . Estimated RIs markedly varied from the literature based RIs that are used in the laboratory.Reference intervals were calculated for: RBC count (4.2-5.4 x10Indirect method employed in this study enables straightforward assessment of RIs in pre-school children. Herein derived RIs differed from the literature-based ones, indicating the need for intra-laboratory determination of RIs for specific populations and sample types. Defining intra-laboratory reference intervals (RIs) for biochemical and haematological parameters is highly important for accurate interpretation of laboratory data. According to the Clinical and Laboratory Standards Institute/International Federation of Clinical Chemistry and Laboratory Medicine (CLSI/IFCC) recommendations, the best procedure for defining RIs is the direct approach, which comprises selection of adequate reference population, sampling, laboratory measurements and statistical analysis of the data , as a part of routine pre-school testing. The used results were collected from three pre-school generations. Sampling was performed in the following periods: February - April 2015 (105 samples), February – April 2016 (97 samples) and January – March 2017 (98 samples).All children underwent general medical examination prior to sampling and were free of any acute or chronic local or systemic disease. The entire study was conducted according to the ethical principles established by the Helsinki Declaration. The study protocol was approved by the Ethics Committee of the Healthcare Centre Kanjiža.2-EDTA . The analysed parameters comprised total blood count: white blood cells (WBC) count; WBC differential - neutrophils, monocytes and lymphocytes counts and percentages; red blood cells (RBC) count; concentration of haemoglobin; haematocrit (HCT); RBC indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cell distribution width (RDW), RDW-standard deviation (RDW-SD); platelets (PLT) count; platelets indices: mean platelet volume (MPV) and platelet distribution width (PDW).Blood sampling was performed in accordance with the current CLSI guidelines. In short, 500 μL of capillary blood was taken between 7 and 8 am by fingerstick, using BD Microtainer contact-activated lancet and BD Microtainer blood collection tubes containing KAll analyses were performed immediately after blood sampling by Sysmex XP-300 automated haematology analyser . Red blood cells, WBC and PLT counts were determined by the direct current detection method with coincidence correction. Populations of cells were separated by automatic discriminators based on complex algorithms. Haematocrit is determined according to the RBC count and volume detection of each individual RBC. Haemoglobin concentration was assessed by the non-cyanide method. All analyses were done simultaneously with three levels  of quality control samples  in each batch of run.Normality of data for each of the examined parameters was tested by using the Kolmogorov-Smirnov test. In case of skewed data distribution, log-normal transformation was applied and data distribution was tested again. The analysed data are presented as mean ± standard deviation for normally distributed variables, as geometric mean  for variables with log-normal distribution, or as median (interquartile range) for variables with skewed distribution. Gender differences between the measured values of included haematological parameters were estimated by the Student t-test for normal data distribution, or the Mann-Whitney U test for skewed data distribution.th and 97.5th percentiles with 90% confidence intervals. For skewed variables and parameters that significantly differed between boys and girls, gender-specific RIs were calculated.Determination of RIs by the indirect approach was assessed by using statistical procedures that were recommended by the IFCC Committee on Reference Intervals and Decision Limits and previously described in detail . Significant differences are considered for P < 0.05.This study included results of haematological parameters determination in capillary blood of 300 pre-school children . The results of data distribution testing are presented in In case of haematological parameters which did not differ between sexes, we used the entire dataset for calculations of gender non-specific RIs. The estimated indirect RIs for RBC count, WBC count and PLT-related parameters are presented in Due to preliminarily observed differences between boys and girls, gender specific RIs were established for number and percentage of monocytes, haematocrit, MCV and RDW . The samFinally, we compared the newly calculated RIs with the literature-based RIs that were in use in our Healthcare Centre laboratory’s everyday practice . In compIn this study, we used the indirect method for the assessment of intra-laboratory RIs for 18 haematological parameters in pre-school children. Unique RIs were calculated for those parameters that did not differ between sexes, while gender-specific RIs were derived in case of parameters that significantly varied among boys and girls. Defining internal laboratory RIs is a demanding procedure in terms of both time and resources. Thus, many routine laboratories preferably use externally derived RIs, although such approach might be associated with errors and misinterpretation of the obtained laboratory results (The main source of errors during the indirect assessment of RIs is related to the selection of an adequate database. The principal goal in this process is to reduce potential inclusion of samples affected by any disease, or a condition that could influence definition of RIs. Herein we used the results obtained from healthy outpatients who were routinely tested during pre-school medical examinations. The advantages of using the results obtained from apparently healthy subjects are clear, since such approach warrants less interferences related to the presence of inflammation, various diseases, medical treatments, dietary changes and other alterations that are frequently seen in in-hospital patients (Next important issue for the estimation of RIs is sample size. It has been suggested that minimal number of samples for estimation of RIs should be 400 (et al. in a study of paediatric population of Caucasian origin pointed towards higher MCV values in girls starting at age of 4, which is consistent with our findings (Another significant question for the statistical approach in the assessment of RIs is data distribution. Namely, general assumption of the recommended statistical procedures for calculation of RIs is that the analysed data set is normally distributed (An important aspect of our study is that RIs were determined based on samples of capillary blood. It has been shown that variations in haematological markers are present in children and adults depending whether venous or capillary blood is used (To get a better insight into the significance of intra-laboratory developed RIs, we compared the RIs determined in this study with the literature-based ones that are currently in use in our laboratory. Although the estimated deviations between the two categories of RIs ranged 0-20% for majority of cases, our analysis indicated some major differences that went up to 47.5%. Such findings emphasize the need for development of intra-laboratory RIs, in order to minimize the possibility of diagnostic errors due to inadequate reference ranges. The observed discrepancies might arise due to the fact that literature-based RIs were defined for venous blood samples. Thus, the development of intra-laboratory RIs, which are adapted to specific laboratory needs and types of samples, is an optimal approach for improving this aspect of laboratory diagnostics. Moreover, literature-based RIs did not recognize gender-specific variations in several parameters, which we identified as significantly different among sexes. Interestingly, the highest level of disagreement between estimated and literature-based RIs was observed for those parameters that differed between boys and girls, thus confirming the inappropriateness of gender-nonspecific RIs in these cases.It should be mentioned that RIs in this study are determined by using 3-part differential haematology analyser, so only three WBC populations are evaluated. For more comprehensive determination of haematological RIs, 5-part differential haematology analyser should be employed.In conclusion, by using the relatively simple indirect method we obtained adequate intra-laboratory reference ranges for haematological parameters in pre-school children. For parameters that significantly differ among sexes, separate RIs for boys and girls are needed. The observed differences among herein-calculated and literature-based RIs accentuate the need for intra-laboratory estimation of RIs, with respect to the sample type and targeted population. Accurate and reliable RIs for haematology analytes in children of different age and gender are of great significance for diagnostics and monitoring of therapy in this specific population.


Patient-reported outcome measures (PROMs) assess patients’ perspectives on their health status, providing opportunities to improve the quality of care. While PROMs are increasingly used in high-income settings, limited data are available on PROMs use for diabetes and hypertension in low-and middle-income countries (LMICs). This scoping review aimed to determine how PROMs are employed for diabetes and hypertension care in LMICs.We searched PubMed, EMBASE, and ClinicalTrials.gov for English-language studies published between August 2009 and August 2019 that measured at least one PROM related to diabetes or hypertension in LMICs. Full texts of included studies were examined to assess study characteristics, target population, outcome focus, PROMs used, and methods for data collection and reporting.Sixty-eight studies met the inclusion criteria and reported on PROMs for people diagnosed with hypertension and/or diabetes and receiving care in health facilities. Thirty-nine (57%) reported on upper-middle-income countries, 19 (28%) reported on lower-middle-income countries, 4 (6%) reported on low-income countries, and 6 (9%) were multi-country. Most focused on diabetes , while 4 studies focused on hypertension and 4 focused on diabetes/hypertension comorbidity. Outcomes of interest varied; most common were glycemic or blood pressure control (38), health literacy and treatment adherence (27), and acute complications (22). Collectively the studies deployed 55 unique tools to measure patient outcomes. Most common were the Morisky Medication Adherence Scale (7) and EuroQoL-5D-3L (7).PROMs are deployed in LMICs around the world, with greatest reported use in LMICs with an upper-middle-income classification. Diabetes PROMs were more widely deployed in LMICs than hypertension PROMs, suggesting an opportunity to adapt PROMs for hypertension. Future research focusing on standardization and simplification could improve future comparability and adaptability across LMIC contexts. Incorporation into national health information systems would best establish PROMs as a means to reveal the effectiveness of person-centered diabetes and hypertension care. Non-communicable diseases (NCDs) account for 71% of global deaths . The estChronic diseases require person-centeredness and consistent, holistic care to ensure good outcomes , 7. SuchA patient-reported outcome is defined as any report of the status of a patient’s health condition that comes directly from the patient, without interpretation of the patient’s response by a clinician or anyone else. Patient-reported outcome measures (PROMs) describe patients’ perceptions of the benefits that they receive from the health system, including patient views on health outcomes and the quality of services received –10. PROMPROMs and PREMs are increasingly used by clinicians and hospitals to guide clinical decision-making and for public reporting of health system performance . PROMs aThis review followed the Preferred Reporting Items for Systematic Reviews and Meta Analyses—Extension for Scoping Reviews (PRISMA-ScR) guidelines . The proWe included studies conducted in LMICs that were: (1) in English, (2) published in a peer-reviewed journal in the 10 years before August 8, 2019 to reflect the period when PROMs in LMICs began to appear in the published literature, and (3) reported use of at least one PROM or a single financial PREM related to hypertension, diabetes, or both . Among LThe review searched PubMed, EMBASE, and ClinicalTrials.gov for randomized controlled trials through 8 August 2019. The search included three components: (1) a PROMs component and (2) a disease component (diabetes and/or hypertension) and (3) a list of LMICs. Search terms were customized for each electronic database. The full strategy is available as We used Covidence  to manage search results and determine review eligibility. We first merged search results from each database and removed duplicate citations. Two reviewers independently screened titles and abstracts of all search results, retrieved full-text articles for the abstracts that received two votes for inclusion, and independently screened the full texts. Studies identified from ClinicalTrials.gov were identified as potentially eligible following title and abstract review. Associated full-text articles were included if available. Conflicts were resolved through reviewer discussion. A senior reviewer (CN) verified eligibility for inclusion during the full-text review only. The reviewers  extracted data from included articles into Microsoft Excel .PROMs and the financial PREM were divided by disease focus: 1) diabetes, 2) hypertension, and 3) both diabetes and hypertension. Articles were further sub-divided by:Location  regions , 18Study populationStudy designLevel of health facilityTools used, status of tool/questionnaire validation and domains measuredAdministration Method of data collection Frequency of evaluationTechnology use Intended use Our search identified 197 studies from PubMed and 31 studies from Embase that met our study criteria. None of the studies identified through ClinicalTrials.gov met the study criteria. Out of the 228 identified articles reporting PROMs in LMICs, 5 duplicate studies were removed. After screening of titles and abstracts, 119 studies proceeded to full-text review and 68 studies were eligible and included in this review .PROMs have been used in LMICs all over the world. Thirty-one LMIC countries from all six WHO regions are represented in the 68 studies included in this review . Among iThe included studies were published between 2010 and 2019, with 45 studies (66%) published between 2016 and 2019. The majority were cross-sectional studies , followed by prospective cohort studies , both prospective and retrospective cohort studies , qualitative studies , descriptive studies , randomized clinical trials  and case-control studies . Baseline characteristics of the included articles are shown in Study population. Participants with established diabetes were reported in 54 studies (90%). Thirty-two studies (53%) focused on Type 2 diabetes, 3 studies (5%) reported on Type 1 diabetes, and 21 studies (35%) reported on both Type 1 and 2 diabetes. Three studies (5%) included adolescents (>12 years), while the remaining 57/60 (95%) focused on adults older than 18 years. Two studies (3%) enrolled older participants (>55 years). Participants were mainly drawn from tertiary hospitals . Most studies  had sample sizes of 500 or fewer. The inclusion criteria for 34 studies (57%) included the treatment regimen, such as insulin, diet/exercise, oral hypoglycemic agents, or combination therapies. In addition to the inclusion criteria, 15 studies (25%) discussed treatment approaches for responding patients.Patient-reported outcomes. Twenty-seven studies (45%) provided outcome data provided by patients only, while 33 studies (55%) described patient-reported outcomes that had been validated by clinician data. A majority of studies  reported on three or fewer patient-reported outcomes. The most commonly reported outcome was glycemic control , followed by health literacy and treatment adherence , acute complications , chronic complications , quality of life , economic accessibility , psychological wellbeing, diabetic stress and depression , patient satisfaction , self-care efficacy , and health services .Data collection/reporting. Two-thirds of studies  assessed outcomes once. Frequency of follow-up varied for the remaining 20 studies (33%), ranging from one week to two years. All 60 studies were conducted as stand-alone surveys; only one study used routinely collected patient-reported outcomes from existing records. Questionnaires were administered by study staff with chart review in 23 studies (38%) and by clinicians in 4 studies (7%). Questionnaires were self-administered by the patients in 13 studies (22%) while the remaining 20 studies did not specify (33%).Study population. All four hypertension PROMs studies targeted adult populations with an established diagnosis of hypertension. Two studies targeted adults ≥18yrs, one study targeted adults ≥55, and one study targeted adults ≥65 yrs. Inclusion criteria included the use of anti-hypertensive medication in 2/4 studies. PROMs were collected at a single time point in either primary (2/4) or tertiary (2/4) health care facilities.Patient-reported outcomes. Three of the four studies of hypertension patient outcomes collected data from patients only, while one study collected data from patients and validated with clinician data. Health literacy and treatment adherence was the most frequently reported focus, reported in 3/4 studies. The following outcomes each appeared in one study: quality of life, burden of care, patient satisfaction, economic accessibility, affordability of transportation costs, and health services as measured by prior hospitalization/admission.Data collection/reporting. All four studies reported on patient reported outcomes as a primary outcome and used stand-alone surveys. One study reported data collected by a staff-administered survey, one reported on focus group discussions, and two did not specify their data collection method.Study population. All four diabetes and hypertension PROMs studies reported on adult populations. Targeted populations included adults ≥18 years with two studies focusing on adults ≥50 years. The studies focused on patients with an already-established diagnosis for hypertension and/or diabetes. Three of four studies measured use of medication. Two studies collected PROMs at a single time point, one study collected PROMs at baseline and six months, while one study collected PROMs at baseline and every three months for two years.Patient-reported outcomes. Patient-reported outcomes were validated with clinical data in three of the four studies. Health literacy and treatment adherence was the most frequently reported outcome (3 studies) followed by diabetic/hypertensive chronic complications (2 studies), glycemic control (2 studies), blood pressure control (2 studies), diabetic/hypertensive acute events (2 studies), economic accessibility (2 studies), and patient satisfaction (1 study).Data collection/reporting. All four studies collected patient reported outcomes as a primary outcome and the surveys were administered by study staff. One study used face-to-face interviews. The survey design from the other three studies was unspecified.There was great variation on the outcomes reported . OverallStudies reported a combined total of 55 unique tools to collect PROMS. Most tools focused on diabetes alone , while four tools focused on hypertension and two tools were used for both hypertension and diabetes . One stuTwo main types of PROMs were utilized. generic and disease-specific. Nineteen (35%) of the 55 tools were specific to diabetes, while the remainder were generic for use across many conditions. Forty-six (84%) of the 55 tools have been validated in English; studies reported extensive translation for use in multiple other languages (anguages . Many toSeventeen out of 68 studies (25%) reported on an economic accessibility PREM. Economic accessibility was assessed primarily by measuring the lack of health insurance coverage, financial barriers to access services, and out-of-pocket expenditures leading to impoverishment.While we did not find studies that evaluated how PROMs were being used in routine care, the authors proposed how their findings would influence the use of PROMs for clinical or policy decision-making. They recommended using PROMs to identify patients who did not meet treatment targets or who reported low treatment satisfaction , 107. PRSome of the barriers noted by authors in the use of PROMs were related to the types of questions including the accuracy of self-reported measures , 41, 116To our knowledge, this scoping review is the first to shed light on the use of PROMs related to diabetes and hypertension in LMICs. We found that PROMs for diabetes and hypertension are being used in every region of the world, and more in upper-middle income countries than in low-income countries. Reported PROM use has increased over time.An emphasis on improving healthcare quality, especially in the context of universal health coverage, may have raised the profile of PROMs. Increased attention to PROMs in high-income countries may translate to greater focus on PROMs in LMICs, particularly given the use of PROMs for clinical decision-making and policies.PROMs have been successfully used to improve quality of care of chronic diseases in high-income countries. Mirroring this impact in LMICs will require appropriate contextual adaptation. PROMs should be validated in LMICs. Further work on translation to other languages would increase accessibility and applicability in LMICs.PROMs can be used to create a feedback loop between providers and patients by identifying patient concerns and addressing system-level factors to improve health outcomes. At a facility level, a short, standardized validated questionnaire could be used as part of routine clinical practice to improve day-to-day patient care. PROMs data could be used locally and aggregated at subnational and national levels. In addition, PROMs can be included in national population-based surveys on diabetes and hypertension. At the regional and national levels, structured, regular PROMs assessment can track progress and inform benchmarking.Simplifying use can facilitate increased adoption. Future research could focus on testing and validating short, simplified, and generic PROMs in LMICs that could be used across multiple NCDs and easily incorporated into routine health systems to allow for standardization and monitoring of trends as well as comparisons between individuals, health facilities, and across countries. In addition, a review of PROMs psychometric properties may be warranted to ensure that they are appropriate to the context.Studies reported a diverse range of patient-reported outcomes. Most studies from low-income countries reported on health literacy and treatment adherence, while disease control and acute complications were the most common focus in middle-income countries. Disease control monitoring requires costly tests, such as HbA1c, which is more commonly measured in upper-middle-income countries. This contrasts with the reliance of many low-income countries on the more affordable approach of blood glucose measurement. Low-income countries may also need to address pressing needs related to infectious disease and maternal and child health, reducing emphasis on non-communicable diseases. Such constraints may lead low-income country health ministries to prioritize measurement of service access over disease outcomes, as this is more fully within health system control. Survival was not included as an outcome because included studies focused on patient-reported data. However, survival would be an outcome of interest for longitudinal studies as well as routine surveillance systems following PROMs over time.High systolic blood pressure is estimated to be seven times more prevalent than diabetes in LMICs, yet most included studies focused on diabetes . ChronicWe identified only four studies that reported PROMs use for diabetes and hypertension comorbidity, based in Mexico (2), Brazil (1), and China (1). High comorbidity and disease burden may have motivated this interest in these three upper middle-income countries where hypertension and diabetes prevalence are above the respective global averages of 31% and 9.3% . Since POur study had several limitations. Although our search strategy was comprehensive, we may have omitted relevant publications not available in the English language or not indexed in PubMed, Embase or ClinicalTrials.gov. Limiting our study search to LMICs constrained our ability to comment on the scale and focus of PROMs in high-income countries. Authors were not consistent in reporting the type of tool used, modes of administration, version, content, and language, revealing variation in the quality of methodologies used. We therefore did not have sufficient data to provide the specific content measured by each tool in each instance of its use. It is possible that hypertension patient outcomes are being measured or described differently from the language used in this scoping review search strategy, leading to the absence of identified hypertension-specific tools. Our focus on patient-reported outcomes meant that we excluded studies that captured only clinical data. As a result, disease control as an outcome is reported only if it was reported alongside other relevant PROMs. It is therefore likely that disease control is measured more widely in LMICs than was captured in this review. Not all LMICs using PROMs may have published their practice in peer-reviewed journals. Finally, our study was limited to PROMs and one PREM related to economic access. We did not collect data on other PREMs, such as access to services. It is possible that low-income countries collect more data on affordability and geographical access than patient-reported outcomes in order to address service provision and patient experience.This scoping review provides a comprehensive overview of where, how, and what PROMs for diabetes and hypertension are being used in LMICs. PROMs are increasingly used all over the world, although less widely in low-income countries than in middle-income countries. Future research should address how PROMs can be incorporated into routine health systems while addressing various challenges, including inconsistencies in administration and specific patient-reported outcomes collected, paper-based data management systems, and resources for tool translation and validation. Development of a simple universal tool with a minimum of key elements that are reported by all patients could reduce costs, allow for incorporation into existing data systems, and facilitate cross-country and cross-condition comparisons. This ongoing tracking could be augmented by periodic in-depth surveys. PROMs provide an exciting opportunity to encourage person-centered, high-quality care.S1 Checklist(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe.Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages  achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages  may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion.Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.Video abstract.The online version contains supplementary material available at 10.1186/s40168-020-00974-y. Microbial phylogeography is the study of the diversification and distribution of microorganisms across space and time, and offers insights into eco-evolutionary processes that generate and maintain the ubiquity and diversity of microbial populations. However, our understanding of microbial phylogeography is far behind that of macroorganisms  and has Pelagibacter   and 23Srhttp://www.R-project.org/), which is a pipeline for assigning reads to single nucleotide-resolved amplicon sequence variants (ASVs). We followed the workflow previously designed for CCS-based long-read amplicon sequencing [The 159,262 CCS reads (average length = 1951 bp) obtained from the 24 samples were analyzed using the DADA2 v. 1.12.1 package  with thequencing : The demThe 742 ASV sequences thus obtained (including both 16S rRNA gene and ITS) were aligned against the SILVA SSU 132 Ref NR database  using thFor each OTU, the pairwise Bray–Curtis dissimilarities of ASV composition were calculated among samples using the vegan v. 2.5-2 package  of the RTo evaluate the performance of our single nucleotide-resolved analysis, we tested whether the single nucleotide polymorphisms (SNPs) predicted from ASVs can be reproduced in shotgun metagenomic reads. We focused on sequences of the CL500-11 lineage in Lake Biwa, and used the assembly and read mapping results generated from a sample collected in the hypolimnion of the lake in September 2016 . Reads mTwo major challenges are associated with the long-read sequencing platform: read accuracy and read throughput. In response to the former challenge, we targeted the 16S rRNA gene and ITS sequences but excluded the adjacent 23S rRNA gene, as a longer insert would result in (i) lower per-base quality of CCS due to fewer rounds of subreads and (ii) a smaller number of error-free reads at the same per-base error rate. To test the accuracy and sensitivity of our analysis, we focused on the CL500-11 lineage in the hypolimnion of Lake Biwa—the OTU and sample with the greatest number of reads (2606 reads) in the present study —and inveRelatively low read throughput is another major challenge facing long-read amplicon sequencing. This limitation occurs because a significant number of reads are discarded during read quality control along with the lower per-cost sequencing throughput compared to short-read sequencing. In the present study, we used the stringent quality filtration threshold proposed for the original DADA2 pipeline developed for CCS-based long-read amplicon sequencing . As a reThe overall community composition in our study showed severe discrepancies compared to that described in a previous study  based onOverall, our analysis generated 742 ASVs assigned to 441 SSU-ASVs and 155 OTUs . The resEach of the dominant lineages harbored 5–34 SSU-ASVs and 7–101 ASVs . DespiteClusters that were separated between Japan and Europe, as well as between Hokkaido and Honshu islands in Japan, were observed in most individual lineages Fig. , indicatPolynucleobacter [Limnohabitans [The existence of epilimnion- and hypolimnion-specific clusters rather than distance–decay relationships among lakes in Honshu and Kyushu Fig.  suggestseobacter , 41 and habitans , 70.Previous studies have investigated temporal trends in the intra-lineage population composition of freshwater bacterioplankton, demonstrating that the community consists of both persistent and transient populations , 71, 72.Ca. Profundisolitarius”), a cosmopolitan bacterioplankton lineage that was dominant in the oxygenated hypolimnia of deep freshwater lakes [In addition to the overall phylogeographic pattern, analyses of individual lineages provided a more detailed perspective on their microdiversification. Here, we focus on CL500-11  clusters exist within CL500-11: MAGs from Lake Biwa, Lake Michigan, and the Caspian Sea were distinct (ANI < 95%) from each other, while those from Lake Zurich were found in both the Lake Michigan and Caspian Sea clusters . The fin% clusterCa. Planktophila versatilis” and “Ca. Planktophila dulcis”) [In contrast to the limited diversification of CL500-11 among Japanese lakes, Iluma-A2 and acI-A1 represented the opposite extreme, as their population compositions were unique for most lakes Figs. , and S3.dulcis”) . The micdulcis”) , 41, 42 dulcis”) .Finally, we note that genomic variation can exist even among cells with identical ITS sequences , 83, 84.In the present study, we applied single nucleotide-resolved long-read amplicon sequencing analysis to a large collection of environmental samples obtained from 11 deep freshwater lakes in Japan and Europe. The results demonstrated sympatric, allopatric, and temporal microdiversity in lake bacterioplankton and revealed phylogeographic patterns that could not be observed based on short-read amplicon sequencing or the MAG-based approach. Whereas previous studies have reported genomes sharing ANI > 95% in freshwater habitats thousands of kilometers apart , 44, 46 Additional file 1:Supplementary Table S1. Detailed profiles of the lakes sampled in the present study. Data were collected from Okazaki et al. [i et al.  and Okazi et al. . † SamplAdditional file 2:Supplementary Table S2. Comparison of the 23Sr  and 23Sr-mod (modified for the present study) primers, showing their coverage for each phylum. Coverage was determined using the TestProbe 3.0 tool with reference to the SILVA LSU 132 Parc database  of reads generated using long-read (this study) and short-read [Figure S2. The relative abundance of ASVs in each sample for each lineage. Rows and columns are clustered based on the Bray-Curtis dissimilarity among samples and ASVs, respectively . Abbreviations for sample names follow those in Fig. Figure S3. Clustering of samples based on Bray–Curtis dissimilarity of amplicon sequence variant composition for each lineage. Abbreviations for sample names follow those in Fig. Figure S4. Comparison of the five most abundant amplicon sequence variants (ASVs) between temporal replicates (2010 and 2015) collected in Lake Biwa for each water layer. Bars indicate the relative abundances of ASVs within each lineage and are ordered by abundance rank for each sample. Gray lines indicate succession of ranks between two time points; N.D., not detected. Supplementary Text (R script).ort-read  platformAdditional file 4:Supplementary Dataset. Summarized dataset related to amplicon sequence variants (ASVs) and operational taxonomic units (OTUs). The Excel file consists of two worksheets. In the first sheet (named “by_ASV”), each row represents an individual ASV. The columns indicate the corresponding SSU-ASV and OTU IDs, the taxonomy assigned to the OTU, and read abundance in each sample. The nucleotide sequences of the ASVs are shown in the last column. In the second sheet (named “by_OTU”), each row represents an individual OTU. The columns indicate the taxonomy, classification based on freshwater bacterioplankton nomenclature (see Methods for details), the number of SSU-ASVs and ASVs assigned to the OTU, the number of samples in which the OTU was detected, and read abundances in total and in each sample. OTUs are sorted based on the total read number. Representative 16S rRNA gene sequences of the OTUs are shown in the last column. In each sheet, the total number of the reads assigned to each sample is shown in the first row. Abbreviations for sample names follow those in Fig.


The Goodpasture syndrome is an extremely rare disease, with renal and pulmonary manifestations, and is mediated by anti-glomerular basement membrane (anti-GBM) antibodies. Renal pathological changes are mainly characterized by glomerular crescent formation and linear immunofluorescent staining for immunoglobulin G on the GBM. There are few reports on the atypical course of the syndrome involving serum-negative anti-GBM antibodies. Therefore, we present a case of Goodpasture syndrome that presented with nephrotic-range proteinuria and was seronegative for anti-GBM antibodies.A 38-year-old Chinese man presented with a lung lesion that was discovered by physical examination a month prior to presentation. The chief concern was occasional hemoptysis without fever, cough, chest pain, and edema.Laboratory testing revealed that the urinary protein level and urine erythrocyte count were 7.4 g/24 hours and 144/high-power field (HPF), respectively. Serological testing for anti-GBM antibodies was negative. Chest computed tomography revealed multiple exudative lesions in both lungs, indicating alveolar infiltration and hemorrhage. Electronic bronchoscopy and pathological examination of the alveolar lavage fluid indicated no abnormalities. However, kidney biopsy suggested cellular crescent formation and segmental necrosis of the globuli, with linear IgG and complement C3 deposition on the GBM. These findings were consistent with the diagnosis of anti-GBM antibody nephritis.The patient underwent 7 sessions of double filtration plasmapheresis. He was also administered with intravenous methylprednisolone and cyclophosphamide. After renal function stabilization, he was discharged under an immunosuppressive regimen comprising of glucocorticoids and cyclophosphamides.Three months later, follow-up examination revealed that the 24-hour urine protein had increased to 13 g. Furthermore, the urine erythrocyte count was 243/HPF. After a 6-month follow-up, the patient achieved partial remission, with a proteinuria level of 3.9 g/24 hours and a urine erythrocyte count of 187/HPF.This extremely rare case of Goodpasture syndrome manifested with seronegativity for anti-GBM antibodies and nephrotic-range proteinuria. Our findings emphasize the importance of renal biopsy for the clinical diagnosis of atypical cases. Furthermore, because renal involvement achieved only partial remission despite therapy, early detection and active treatment of the Goodpasture syndrome is necessary to improve the prognosis of patients. The discovery of anti-GBM antibodies in 1967 verified the pathogenesis of the Goodpasture syndrome. However, only few studies have investigated the atypical course of the syndrome involving serum-negative anti-GBM antibodies. We present a case of Goodpasture syndrome with serology negative for anti-GBM antibodies and manifested as nephrotic-range proteinuria. The purpose of this report is to put forward new reflections for clinicians regarding this attractive case.The Goodpasture syndrome is a rare autoimmune disease that is mediated by anti-glomerular basement membrane (anti-GBM) antibodies. Acute kidney failure and life-threatening pulmonary hemorrhage are typical clinical symptoms.22, 40.7 g/L, 7.4 g/24 hours, and 144/HPF  scan indicated multiple exudative lesions in both lungs, indicating alveolar infiltration and hemorrhage . At the last follow-up (6th month), the serum albumin, serum creatinine level, proteinuria level, urine erythrocyte count, and eGFR were 40 g/L, 77 μmol/L, 3.9 g/d, 187/HPF, and 108.7 ml/minute/1.73 mm2, respectively. The patient provided informed consent for the inclusion of his data in the present report.Based on the renal pathology and laboratory findings, double filtration plasmapheresis , pulse methylprednisolone therapy (10 mg/kg daily for 3 consecutive days), and pulse cyclophosphamide treatment (1 g) were initiated. Simultaneously, entecavir was administrated to reduce HBV replication. Following DFPP, chest CT was repeated, which revealed that the pulmonary lesion had almost disappeared  chain is mainly expressed in a few specialized basement membranes like the GBM. The anti-GBM antibody binds to the a3(IV) NC1 domain and deposits in the basement membranes of the kidneys and the lungs, subsequently activating the complement system that can result in tissue damage. Although the pathogenesis of the disease in the kidneys and the lungs is the same, the degree of damage is markedly different between them. It was reported that two-thirds of the patients with anti-GBM antibodies could progress to the Goodpasture syndrome, and one-third present with anti-GBM antibody nephritis without pulmonary manifestations.,7 Clinically, enzyme-linked immunosorbent assay (ELISA) is a common method for the detection of anti-GBM antibodies, especially if a kidney biopsy is not performed, and has high sensitivity (>95%) and specificity (>97%). However, as previously reported, certain cases of the Goodpasture syndrome are seronegative for anti-GBM antibodies, and the reasons for the same have not been elucidated.The disease is caused by anti-GBM antibodies targeted against the non-collagenous domain (NC1) of the alpha 3 chain (a3) of type IV collagen in the lungs and kidneys. however, one of the key characteristics of our report is the nephrotic-range proteinuria without renal dysfunction, which indicates a more severe degree of podocyte injury as compared to in the classic form. Nasr et al recently reported 7 patients who presented with nephrotic syndromes presumed to be atypical GBM diseases; however, the presence or absence of serum anti-GBM antibodies was not described in these cases. Liang et al also reviewed the clinical pathological features of antibody-negative anti-GBM disease, and observed that 7 out of 19 patients presented with nephrotic-range proteinuria; however, it is unclear whether the kidney function was impaired. Therefore, we believe that our report, which details a case seronegative for anti-GBM antibodies and presenting with nephrotic-range proteinuria without kidney failure, is fairly rare.In our case, the patient was seronegative for ANCA and anti-GBM antibodies, and presented with the Goodpasture syndrome with nephrotic-range proteinuria and normal renal function. While this condition may clinically be misdiagnosed as membranous nephropathy and membranous proliferative glomerulonephritis, among others, renal pathology revealed crescent formation and necrotic changes, thereby enabling a confirmative diagnosis. Immunofluorescence staining also indicated a linear deposition of immunoglobulin G and complement C3 on the GBM. Considering that the impairment of the lungs was due to alveolar hemorrhage, the patient was diagnosed with atypical Goodpasture syndrome. There are 2 relatively rare aspects of our case. Usually, classical anti-GBM disease presents with rapidly progressive kidney failure;; this demonstrates that renal tissues may have different epitopes than those present in the native kidney. Secondly, the seronegativity for anti-GBM antibodies may be attributed to their shorter half-life in circulation than in the renal tissue; the antibodies may have disappeared from the circulation while collecting the blood during testing. Finally, antibodies with high affinity are removed from the patient's plasma by binding to the kidney and the alveolar basement membranes. Conversely, the circulating serum antibodies may have a lower affinity than those in the renal and lung tissues; therefore, these may have washed off more easily during the experimental rinsing step. Hence, it is rather important to perform a renal biopsy in highly suspicious patients that are seronegative for anti-GBM antibodies, even though the clinical manifestations are atypical.The mechanism behind undetectable circulating anti-GBM antibodies remains unclear; although there may be several explanations to this. Firstly, it is possible that the result was falsely negative due to the limitations of the test. Even though ELISA has a positive predictive value of 95%, antibody detection remains difficult in a small number of patients. It was reported the auto-antibody antigens included the α3NC1–α5NC1 domains and the linear type IV collagen epitopesThe classical treatment regimens for the Goodpasture syndrome include plasma exchange, corticosteroids, and immunosuppressive therapy. Plasma exchange could eliminate the circulating antibodies. Corticosteroids and immunosuppressants could prevent the formation of anti-GBM antibodies. In our case, the patient received plasma exchange therapy, intravenous methylprednisolone , and cyclophosphamide. These treatments relieved the lung lesion remarkably and ceased hemoptysis. However, only partial remission was achieved in case of renal involvement. The renal function was preserved and the proteinuria level was decreased by more than 50%. Considering that the Goodpasture syndrome could also lead to podocytopathy, tacrolimus was chosen for maintenance treatment. Our observations indicate that Goodpasture syndrome with nephrotic-range proteinuria may require a longer therapy and follow-up to determine prognosis.In conclusion, we have reported the case of a patient who was diagnosed with antibody-negative Goodpasture syndrome accompanied by nephrotic-range proteinuria and normal kidney function. This presentation is different from that of classical cases. This report serves as a reminder for clinicians to maintain a high degree of awareness for identifying such uncommon cases.Conceptualization: Wei Qin.Data curation: ZhengXia Zhong, JiaXing Tan.Investigation: ZhengXia Zhong.Project administration: ZhengFu Li.Writing – original draft: ZhengXia Zhong.Writing – review & editing: Yi Tang Wei Qin.


The medical records of 270 locally advanced cervical cancer patients who were treated with CCRT were collected from three institutions and reviewed retrospectively. A nomogram was used for predicting 2-year disease-free survival (DFS) and 5-year overall survival (OS) based on Cox proportional hazards regression. Predictor variables included nodal maximum standardized uptake value (SUVmax), primary tumor SUVmax, age, tumor size, stage, serum squamous cell carcinoma antigen level, and human papillomavirus status. Internal nomogram validation was performed. A nomogram for predicting the 2-year DFS and 5-year OS was constructed using six and seven parameters, respectively. With a focus on 2-year DFS, our model found nodal SUVmax to be the highest weighted negative prognostic factor. With a focus on 5-year OS, young age was the highest weighted negative prognostic factor. The concordance index was 0.75 and 0.78 for the 2-year DFS and 5-year OS, respectively. This nomogram is a predictive tool that can be used to counsel patients for predicting survival outcomes. Moreover, our prognosis-predicting model may make it possible to personalize treatment.This study aimed to develop a prognosis-predicting model based on [ For locally advanced cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IV], concurrent chemoradiotherapy (CCRT) using a cisplatin-based regimen is currently the standard treatment modality ,2. The c18F]fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) for the prediction of tumor recurrence in locally advanced cervical cancer /body weight [g]). All FDG PET/CT imaging data were transferred to the image archive server  using the Digital Imaging and Communications in Medicine standard. All images were centrally reviewed by two nuclear medicine physicians using the volume viewer software on an Advantage Workstation 4.5 , which provides a convenient and automatic method to delineate the volume of interest using an isocontour threshold method based on SUV. For semiquantitative analysis, SUVmax was designated as the highest SUV of the primary tumor and regional lymph nodes. SUVmax was obtained using the following formula: Clinical follow-ups of patients were performed every 3 months for 2 years, every 6 months between 2 and 5 years, and annually thereafter. Failure was defined as biopsy-proven recurrence or documentation of disease progression on serial imaging studies. The number of recurrent patients and the recurrence rate were calculated for each independent variable level. The Contal and O’Quigley technique was used to select the cutoff value for each continuous independent variable. The estimate of 2-year disease-free survival DFS, 5-year overall survival (OS) and 95% confidence interval (CI) for each independent variable level was determined using the Kaplan–Meier estimator, and the statistical significance was tested using the log-rank test for each independent variable. The crude proportional hazard rate (HR) of 2-year DFS and 5-year OS for each independent variable was estimated using the univariate and multivariate Cox proportional-hazards model (PHM) for the adjusted HR. To calculate conditional survival probability intuitively, nomogram of the 2-year DFS and 5-year OS prediction models based on a cox’s PHM was used, as described previously . To findp-value was less than 0.05.Survival analyses was performed using SAS version 9.4  and the figure plotting was performed using the rms package of R version 3.5.2 for windows. Data were determined to be statistically significant if the The nomogram’s predictive accuracy was measured by the concordance index, which quantifies the level of concordance between predicted probabilities and the actual chance of recurrence or death. This was calculated by bootstrapping the samples from the original 270 patients used to fit the Cox model and served as an unbiased measure of the ability of the nomogram to discriminate patients. The nomogram was calibrated by grouping patients with respect to their nomogram-predicted probabilities and comparing group means with observed Kaplan–Meier estimates for DFS and OS. Bootstrapping was then repeated 200 times.Clinicopathological and PET metabolic parameters of the model derivation cohort. The characteristics of the 270 enrolled patients are summarized in Multivariate Cox proportional-hazards model was used to evaluate independent prognostic factors and estimate their effect on DFS and OS. Six variables were identified as independent risk factors for DFS; these included age, FIGO stage, tumor size, serum SCC antigen levels, pSUVmax, and nSUVmax . VariablA nomogram was constructed based on these six and seven independent risk factors for 2-year DFS and 5-year OS, respectively. In the nomogram, the point of each risk factor was identified on the top scale, and the total points were calculated by totaling the points of the risk factors. The probability of mortality can be assigned by applying the total points to the bottom scale of nomogram . The totThe calibration plots for the probability of 2-year DFS and 5-year OS demonstrated optimal agreement between prediction by the nomogram and actual observation. The calibration curve and concordance for the nomogram are illustrated in p < 0.0001, The discrimination ability of the nomogram in predicting DFS and OS was analyzed by dividing the predicted probabilities of survival outcomes in two groups that were then used to plot the Kaplan–Meier curve. Optimal cutoff values were determined by an algorithm for the maximization of hazard ratio to divide the high- and low-risk groups. The nomogram could stratify patients into low- and high-risk subgroups, and cut-off scores for DFS and OS were 102 and 214, respectively. Furthermore, DFS and OS were significantly higher in the low-risk group than in the high-risk group  between the independent variables were all less than 1.2, and there were no multiple collinearity problems. In this study, we developed and validated a nomogram based on the 18F-FDG PET/CT to the prognostic nomogram for locally advanced cervical cancer patients treated with CCRT [18F-FDG PET lymph node status, cervical tumor SUVmax, and PET tumor volume combined in a nomogram created a good model for predicting recurrence-free survival and OS [A nomogram is a predictive tool that generates the numerical probability of a clinical event by creating a simple graphical representation of a statistical predictive model . Severalith CCRT . In thisl and OS . 18F-FDG PET/CT metabolic parameters for predicting tumor recurrence in locally advanced cervical cancer treated with CCRT. Among SUVmax, metabolic tumor volume, total lesion glycolysis of the primary tumor, regional lymph node, and whole body, and tumor heterogeneity, the SUVmax of regional lymph node was the most powerful biomarker for predicting tumor recurrence in locally advanced cervical cancer treated with CCRT [18F-FDG PET-based prognostic nomograms included lymph node status as a prognostic factor; however, only lymph node level was included [18F-FDG PET/CT metabolic parameters and clinicopathological parameters [Previously, we evaluated the ith CCRT ,13. Previncluded . Moreoveincluded . In thisincluded ,13. Withincluded ,26,27. Crameters . 18F-FDG PET, there may be sampling bias. Second, three different PET/CT scanners were used. Thus, there may have been some differences between institutions with regard to image acquisition and reconstruction. To minimize these differences, reconstruction processing and imaging analyses were performed at a single institution (KNUCH) using the same software (Advantage Workstation 4.5 software) for all calculations. Third, external validation was not performed.Our study has some noteworthy limitations. First, it is a retrospective study. The possibility of selection bias exists. Because this study was conducted only on patients who underwent 18F-FDG PET/CT metabolic and clinicopathologic factors with 270 cases of locally advanced cervical cancer treated with CCRT. Moreover, our prognosis-predicting model is the first nomogram that combines 18F-FDG PET/CT metabolic and clinicopathological parameters. Despites these limitations, our study offers some unique and significant findings and it differs from previous studies in several aspects. This study is the largest study so far, to develop a prognostic nomogram based on pretreatment 18F-FDG PET/CT and clinicopathological characteristics could be used for the homogenization of various prognostic nomograms.In conclusion, we developed and internally validated a risk assessment model using a nomogram for predicting the probability of 2-year DFS and 5-year OS after CCRT in locally advanced cervical cancer. This nomogram may be helpful in identifying patients with high risk of recurrence or compromised survival and allow physicians to choose appropriate adjuvant treatments, such as hysterectomy or chemotherapy, and counsel patients. Moreover, our prognostic variables offered by a nomogram based on


Objective: To explore preliminary effectiveness of the Cognitive Orientation to daily Occupational Performance (CO-OP) Approach in improving outcomes in childhood-onset hyperkinetic movement disorders (HMDs) including dyskinetic cerebral palsy following deep brain stimulation (DBS) across UK clinical occupational therapists.Methods: Randomized, multiple-baseline, Single Case Experimental Design N-of-1 trial with replications across participants. Five self-selected goals were identified: three goals were worked on during CO-OP and two goals were left untreated and used to assess skills transfer. Participants were between 6 and 21 years and had received DBS surgery with baseline Manual Ability Classification System (MACS) levels I–IV. Participants were randomized to typical or extended baseline (2 vs. 6 weeks), followed by 10 weekly individual CO-OP sessions. The primary outcome was functional performance measured by the Performance Quality Rating Scale-Individualized (PQRS-I), assessed before, during, and following treatment. Outcome assessors were blinded to baseline allocation, session number, and assessment time. A non-overlapping index, Tau-U, was used to measure effect size.Results: Of the 12 participants recruited, 10 commenced and completed treatment. In total, 63% of trained goals improved with effect sizes 0.66–1.00 (“moderate” to “large” effect), seen for all children in at least one goal. Skills transfer was found in 37% of the untrained goals in six participants.Conclusions: Cognitive strategy use improved participant-selected functional goals in childhood-onset HMD, more than just practice during baseline. Preliminary effectiveness is shown when the intervention is delivered in clinical practice by different therapists in routine clinical settings. In children and young people with a hyperkinetic movement disorder (HMD) including dyskinetic cerebral palsy (CP) surgical treatments such as deep brain stimulation (DBS) can be effective in reducing severity of motor impairments  , 2. HoweA recent systematic review of interventions for CP indicated strong evidence that surgical interventions, such as intrathecal baclofen infusion pump or selective dorsal rhizotomy, and medical interventions, such as botulinum toxin injections, reduce tone in children with spastic CP . HoweverNevertheless, rehabilitation approaches are available but, to date, lack robust evidence for their feasibility and efficacy. We have recently reported a proof-of-concept efficacy study  of the CTherapist effects, including training and experience, can be a fundamental variable in studies investigating any rehabilitation intervention effectiveness –11. SingThis study aimed to explore whether the results from a previous single-case experimental design proof-of-concept (efficacy) study , 14 coulThe study evaluated the CO-OP approach, as an adjunct to DBS, delivered by non-specialist occupational therapists with a range of clinical experience, to children and young people with HMD. The full trial protocol is available  and was Does the CO-OP approach improve outcomes for children and young people with HMD on participant-selected goals when undertaken in a non-specialized clinical setting?Ethical approval was obtained by the NHS Health Research Authority . The trial was registered (ISRCTN57997252). Written informed consent was obtained from all parents and from participants over 12 years of age. Assent was obtained from younger children. Written permission was obtained in all cases.This study used a randomized, multiple-baseline, N-of-1 design with replications across six therapists (N-of-1 with five replications) in 12 children and young people  (NB: number achieved was N-of-1 with 9 replications across participants).A consecutive series of multiple baseline N-of-1 trials was completed using concealed randomization to allocate length of baseline (2 vs. 6 weeks). The extended baseline permitted an examination of whether repeated baselining  and DBS only has an impact on skill improvement.In an N-of-1 design with replications across participants, sample (series) size is not based on the power to test group effects using inferential statistics. Instead, each separate trial examines change over time within an individual, thus allowing us to determine whether treatment is effective for each individual using predefined quantitative and qualitative criteria. In N-of-1 studies, the number of replications (participants recruited) chosen after the first case is often based on pragmatic grounds related to the known heterogeneity of the clinical sample. The number of measurements within each time period is also based on pragmatic factors such as length of treatment and the likely variability in outcome. It is recommended that an initial N-of-1 trial plus three replications are necessary as a minimum to explore efficacy of an intervention but five replications is better . Similarn = 27). The 10 patients that had participated in the previous study were not eligible. Full inclusion criteria for the study has been reported elsewhere (in situ and without signs of infection.The study included children and young people with HMD who had previously undergone DBS (or where surgery was scheduled) at the complex motor disorders service (CMDS) database at Evelina London Children's Hospital, UK, that met the inclusion criteria from information in their medical records n = 2. The 10 Twelve participants were recruited and admitted to the study sequentially. Further information about eligibility criteria and recruitment procedure are outlined in the study protocol , 15. FigThe Evelina Children's Hospital is a tertiary treatment center providing advanced interventions for children with complex motor disorders. Children are referred from across the UK. For this trial, 19 therapists were initially contacted in centers close to the homes of the 27 potential participants, and 3 additional therapists were approached via special interest group networks. Seven agreed to take part in the study prior to enrolment of the first participant, with six eventually matched to one or more patient participants. Details of the therapists are provided in CO-OP intervention comprised 10 treatment sessions of up to 1 h each, delivered weekly at the participant's home. The frequency of intervention sessions was negotiated between the treating therapist and the participant and family. All therapists had attended CO-OP training workshops (2–3 days) led by CO-OP Academy certified instructors. Ongoing clinical supervision was provided by investigator HG. For more information, please see study protocols , 15.Performance change in self (participant)-selected goals was measured using the Performance Quality Rating Scale—individualized (PQRS-i) . This isAssessments of outcomes were completed several times at each study phase . Five task-based goals were self-identified by each participant with the aid of the Canadian Occupational Performance Measure (COPM)   regression, initially fitting a naïve linear regression model using ordinary least squares (OLS) for reference only (U)  for values 0.66 to 0.92, and “weak” when values are ≤0.65.Serial dependency at baseline was firstly calculated using auto-correlation (AC) so that the most appropriate analysis approach could be chosen, which were (i) differences in individual means and 95% confidence interval for overall change pre- and post-nce only , 23; andonly (U)  taking iA change of at least 2 points on the PQRS-i was used to indicate clinical significance in order to match other similar scales such as the COPM . Differet-test) were calculated for those participants randomized to 6 weeks baseline length.Quantitative statistical analysis of performance change between CO-OP and treatment as usual : Changes in performance across the extended baseline (6 weeks with at least 18 data points) was specifically evaluated. Differences between individual goal means and 95% confidence interval for change of first session and sixth session of baseline  ranged in age from 8 years and 6 months to 19 years and 10 months, with a range of primary diagnoses, etiologies, and clinical phenotypes . DuratioResults of participant 1 are presented graphically with trained and untrained goals in Statistical differences between means in baseline and post-intervention supported the visual inspections and indicated that all participants achieved a significant improvement on at least one trained goal, with 63% (19/30) of goals improving across participants at post-test , 3. Two U correction for baseline trend was used. Using this index, all participants achieved improvement in at least one goal with moderate or large effect size. A “large” effect (≥0.93) (“very effective intervention”) was obtained in 80% of children post-intervention. In total, the effect size was “large” for 37% of trained goals overall post-treatment. For other trained goals, “moderate” effects (0.66 to 0.92) (“effective intervention”) were obtained in 70% of children and for 27% of trained goals overall at post-intervention. “Weak” effects (≤0.65) were seen in 11 goals (37%) overall across eight children at post-intervention. No negative effects (deterioration) were observed for trained goals.Baseline trend  was found in six goals and therefore the use of Tau-U.For untrained goals, “large” or “moderate” effect was seen in 37% of goals (7/19) overall in six children post-treatment, and “weak” effects in 53%. Deterioration with “moderate” negative effects were seen in two goals for two different children. All participants showed a positive change of at least two PQRS-i points (based on the difference between the phase means) on at least one goal, at the end of treatment , 3. Postt-test supported the visual inspections and indicated that DBS and practice alone did not offer improvement in the majority of goals . The present study was set up as preliminary evaluation of effectiveness  and reports the results in performance improvement when the intervention is delivered by local occupational therapists not specialized in HMD and with only basic training in the CO-OP approach based in centers across the UK. Together, the two studies provide evidence that CO-OP is a feasible and acceptable intervention for children and young people with HMD following DBS, with the potential to produce clinically meaningful improvement.n = 10) reported here and in combination with those reported previously (N-of-1 plus 8 replications) exceeds the three to five replications recommended in single-case experimental design, increasing confidence in the results. In this study, all eligible participants in the CMDS database were approached and all who consented were recruited to the study, reducing the risk of bias selection.The results obtained in the present study are promising on two grounds: Firstly, although treatment fidelity was variable, CO-OP can be delivered by occupational therapists independent of their years of training and with a relative low-intensity training course, which makes this approach feasible in the context of clinical practice for further formal evaluation. Secondly, replicability has been demonstrated across multiple therapists, in a substantial number of children and young people with heterogeneous presentations of HMD and DBS duration. The total number of successful replications , toothbrushing and drinking for child with metabolic disorder (participant 9), eating with knife and fork, and applying mascara for child with CP (participant 12). Possible explanations for the lack of improvement could relate to the difficulty of the selected goal in relation the participants' motor impairment, the fidelity of treatment, or the expertise of the therapist. Although challenging, similar goals successfully improved in other participants with the same characteristics, suggesting that the goal itself may not be the limiting factor or the clinical characteristics. Therapist effects may have been important in some of the cases that showed limited clinical gains. Fidelity to treatment was rated <50% for case 4 (38%) and case 9 (49%) who both improved on only a single goal. Therefore, wider improvement might have been hindered by the diluted delivered version of the CO-OP intervention. It may well be that outcome relates to a combination of the difficulty in the chosen goal itself and the therapists' difficulty to apply the CO-OP approach to these more challenging goals. Future trials may require greater investment in training and supervision.Although improvement on trained goals is an important outcome, the real potential of this treatment lies in its generalizability and transferability, enabling improving performance beyond the treatment sessions, thereby broadening the reach of the CO-OP approach. In the present study skill transfer was observed in seven of the untrained goals, across five participants. This is similar to the previous single-therapist HMD single-case experimental design  and in ain situ prior to the systematic application of the CO-OP approach. Improvement during extended baseline (DBS and practice) , it is more likely due to practice effects than DBS. In the remainder, who showed stable baseline performance, improvement could be more confidently attributed to the CO-OP approach.Finally, the present study permitted an assessment of the impact of repeated practice on the goals with DBS ractice)  was notein situ, raising the possibility that DBS-related factors may moderate the outcome of CO-OP. The careful design of both, this and the former proof-of-concept study , and any modifications that might be required to achieve transfer to goals not addressed in therapy for this population. Finally, the promising results from using CO-OP with HMD and DBS warrant further investigation of the CO-OP approach in dystonia, which offers a treatment option for a wider number of potential patients. Further, the results from this study and the previous study indicate that practice alone does not provide improvement in self-selected goals. This suggest that simple goal-oriented approaches recommended by experts in dystonia might not be sufficient without the more cognitive problem-solving element inherent in CO-OP. Future research comparing CO-OP with other goal-oriented approaches would be valuable.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Oxford A Research Ethics Committee, 14/SC/1159. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.HG completed all statistical analysis. This was supervised by VC, senior trial statistician. HG: research study, conception, organization, execution, manuscript preparation, and writing of the first draft. HP, J-PL, VC, and RB: research study, conception, manuscript preparation, and review and critique. All authors contributed to the article and approved the submitted version.J-PL has received unrestricted educational support for instructional courses and consultancy fees from Medtronic Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.