DHH-associated 46,XY gonadal dysgenesis (GD) or 46,XY sex reversal 7, GNPTAB-associated mucolipidosis II alpha/beta (ML II), BBS1-associated Bardet–Biedl Syndrome (BBS), SURF1-associated Leigh Syndrome (LS) and AP4B1-associated spastic paraplegia-47 (SPG47) in unrelated affected members from Bangladesh. Our analysis pipeline detected three homozygous mutations, including a novel c. 863 G > C (p.Pro288Arg) variant in DHH, and two compound heterozygous variants, including two novel variants: c.2972dupT (p.Met991Ilefs*) in GNPTAB and c.229 G > C (p.Gly77Arg) in SURF1. All mutations were validated by Sanger sequencing. Collectively, this study adds to the genetic heterogeneity of rare genetic diseases and is the first report elucidating the genetic profile of (consanguineous and nonconsanguineous) rare genetic diseases in the Bangladesh population.Collectively, rare genetic diseases affect a significant number of individuals worldwide. In this study, we have conducted whole-exome sequencing (WES) and identified underlying pathogenic or likely pathogenic variants in five children with rare genetic diseases. We present evidence for disease-causing autosomal recessive variants in a range of disease-associated genes such as  These disorders encompass a spectrum of rare complex clinical manifestations3 that can be difficult to diagnose and pinpoint causation. Clinics in developing countries are not equipped with technologically advanced healthcare facilities for diagnosis and treatment. In Bangladesh, genetic tests are not widely used in a clinical setting and mostly applied for medical research purposes5. The current standard of care does not include first-tier genetic testing in most developing nations. Limited use of targeted (single-gene) sequencing, low-resolution microarray, and karyotype technologies can identify a small fraction of the total population of patients with rare genetic disorders.Rare genetic disorders, while individually uncommon, collectively impact significant numbers worldwide9. Diagnoses of rare genetic diseases are essential for improving the health care system of any developing country. Early disease diagnosis can lead to early and targeted therapeutic intervention and can avoid other unnecessary diagnostic investigations. In this study, we performed WES to investigate a series of patients from Bangladesh with rare genetic diseases referred for clinical assessment. Currently, there is no standard of care for rare disorders in Bangladesh. Clinical presentation in these patients was complex10, prompting referral for genetic diagnosis. We report seven (7) extremely rare recessive variants that are potentially causal in a range of rare genetic diseases such as, DHH-associated 46,XY gonadal dysgenesis (GD) or 46,XY sex reversal 7, GNPTAB-associated mucolipidosis II alpha/beta ((ML II), BBS1-associated Bardet–Biedl Syndrome (BBS), SURF1-associated Leigh Syndrome (LS) and AP4B1-associated spastic paraplegia-47 (SPG47)from a small cohort of five unrelated Bangladeshi patients.Whole exome sequencing (WES) is both an effective and efficient method to detect genetic defects underlying hereditary diseasesDHH, BBS1, and AP4B1 genes, respectively  unrelated patients with extremely rare pediatric genetic diseases carrying autosomal recessive pathogenic variants in different genes. Specifically, WES identified three homozygous variants: c.863 G > C p.Pro288Arg), c.1339 G > A (p.Ala447Thr), and c.1216 C > T (p.Arg406Ter) in 88Arg, c.A 22-year-old girl, presenting with primary amenorrhea and underdevelopment of sexual characteristics, was referred for genetic evaluation. Her antenatal and peripartum history was uneventful. She was born by normal delivery and declared as a girl by a local physician, and raised as a female. Her cognitive, language, and motor developmental milestones were all normal, and her school performance was satisfactory in the mainstream setting. She was the third child of a non-consanguineous marriage, with no known family history of medical illness. At the age of 15 years, she failed to establish menses and consult with a local physician. Later, she was referred to NeuroGen Children’s Healthcare for clinical exome tests by the clinician at the department of Endocrinology & Metabolism, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh at the age of 22 years. At that examination, her height was 164 cm, BMI 17.1 kg/m2, and arm span 186 cm. Tanner staging revealed breast stage B2(Rt) & B3(Lt), pubic hair stage 4 with female pattern, normal female external genitalia with no palpable gonads. Hormonal evaluation showed FSH 175.12 IU/L , LH 51.13 IU/L , serum testosterone 92.8 ng/dl , testosterone after HCG stimulation test 45 ng/dl, TSH 2.31 uIU/ml , estrogen 77.5 pg/ml , ACTH 22.6 pg/ml , cortisol 637 nmol/L. USG of the abdomen showed absence of Mullerian derivatives. MRI of the abdomen revealed a testis in the left inguinal region, and karyotyping showed 46XY.DHH gene was found that causes Proline to Arginine change at position 288 which belongs to DHh-C functional domain (amino acids 199–396) of Desert Hedgehog protein. This variation was not observed in the gnomAD database (http://gnomad.broadinstitute.org) or the Human Gene Mutations Database (HGMD) (http://www.hgmd.org) or the 1000 Genomes Project and most likely reflects a novel mutation within this domain were classified as pathogenic for similar phenotypic patients in ClinVar database. This reported homozygous mutation can be classified as likely pathogenic in accordance with ACMG criteria13. For the remainder of patients, we have used similar criteria for variant interpretations.After performing WES, a homozygous variant c.863 C > G . She presented with craniofacial dysmorphology, including a prominent philtrum, coarse facial features, and widely spaced nipples. Her joint contractures of both knee and elbows were suspected to be the consequence of intrauterine growth restriction (IUGR). She was the firstborn of a non-consanguineous marriage and her paternal and maternal family members were healthy.GNPTAB was identified , the Human Gene Mutations Database (HGMD) (http://www.hgmd.org) or the 1000 Genomes Project, and most likely reflects a novel variant. These novel compound (frameshifts) variants can be classified as ‘likely pathogenic’ as they meet the likely pathogenic ACMG criteria13.Following WES, a compound heterozygous mutation impacting lTC p.Leu168Glnfs*Patient 3, an 8 year-4-month-old boy, was referred to the pediatric neurology clinic due to focal seizures with loss of consciousness occurring 8 times within a 12 months period. His cognitive and language milestones were all delayed, and he also presented with constipation, and obesity. On examination, polydactyly and strabismus were noted, and initial workup revealed hypothyroidism. He was the second born of a consanguineous marriage and his older sister was healthy.BBS1gene was identified using WES (c.1339 G > A[p.Ala447Thr])that causes Alanine to Threonine change at position 447 of BBS1 protein. This gene is a known cause of Bardet–Biedl Syndrome (BBS), an autosomal recessive and genetically heterogeneous ciliopathy characterized by retinitis pigmentosa, obesity, kidney dysfunction, polydactyly, behavioral dysfunction, and hypogonadism18. In very rare cases, seizure has also been found in patients of BBS20. The variant c.1339 G > A falls at the last nucleotide of exon 13 of the BBS1 coding sequence, which is part of the consensus splice site for this exon. Nucleotide substitutions within the consensus splice site are relatively common causes of aberrant splicing21. This variant was not found in the Genome Aggregation Database (gnomAD) as homozygous state. The heterozygous state of the variant is present in population databases at extremely rare frequencies . Patient 3 meets the major clinical features of BBS with regard to hypothyroidism, polydactyly, obesity, strabismus, impaired intelligence in comparison to peers, and language delay. Therefore, this variant can be classified as ‘likely pathogenic’ in accordance with ACMG guideline. This variant was not screened in the parents’ DNA  respectively. CT Scan of his brain identified bilateral cerebral and cerebellum leukodystrophy/ischemic change. He was the second child of a nonconsanguineous marriage and his elder brother was healthy.SURF1gene was identified using WES (c.229 G > C[p.Gly77Arg] andc.792_793delAG[p.Arg264Serfs*27]). Mutations in this gene are a cause of Surf1 protein deficiency, a recessively inherited severe mitochondrial neurological disorder, and is the most frequent cause of Leigh syndrome (LS) associated with cytochrome c oxidase  deficiency23. The maternally inherited heterozygous missense variant c.229 G > C [p.Gly77Arg]  of the Surf1 protein that might disrupt the assembly of the surf1 protein in the mitochondrial inner membrane. This variation was not observed in gnomAD database (http://gnomad.broadinstitute.org) or the Human Gene Mutations Database (HGMD) (http://www.hgmd.org) or the 1000 Genomes Project and likely reflects a novel mutation. In silico analysis tools SIFT_score24, PolyPhen-211, and CADD_phred12 predict that the variant is likely to be pathogenic .A compound heterozygous variant in Patient 5, a 15-month old boy born to consanguineous healthy parents, was referred for genetic testing after being admitted to the hospital with an 8-min tonic-clonic seizure in the context of a 48-h fever. Prior to this, there had been no major concerns. He was delivered by an uneventful LSCS following a normal pregnancy and without any perinatal complications. His birth weight was 2.5 kg. He was able to hold up his head unsupported at 3 months, sit upright independently at 10 months, and by 15 months was able to walk a few steps with support. His speech was delayed, however, and he was only using one and two-syllable words such as “baba”, “mama”, and “dada”. On examination, at the time of first admission, he was conscious, yet irritable, and his vital signs were within the reference range. His muscle bulk was normal, as were muscle tone and reflexes. He had a plantar-extensor response. His EEG showed some non-specific background polymorphic activity. Brain MRI examination showed minimal atrophy in both fronto-parietal and temporal lobes. Serum ammonia and lactic acid level were 60.0 µmol/L and 1.3 mmol/L respectively. He was the second-born, with his older female sibling, now age 6 years, previously diagnosed with psychomotor and language delay.AP4B1-impacting homozygous variant c.1216 C > T (p.Arg406Ter) was identified that alters an amino acid from Arginine to stop codon, p.Arg406Ter.The AP4B1 gene encodes a 739-amino acids beta 1 subunit of the heterotetrameric adaptor protein (AP) complex28. Abnormality of this gene leads to spastic paraplegia-47 (SPG47), intellectual disability, or an AP-4 deficiency syndrome31. The variantc.1216 C > T is predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. It is a very rare allele; the allele frequency of this variant in gnomAD (http://gnomad.broadinstitute.org) is 0.00005567 (14/251462). These 14 alleles are present as heterozygotes and no homozygotes were observed in gnomAD. There is also no entry of this variant in 1000 g database, indicating it is not a common variant. ClinVar27 has two submissions for this variant (Variation ID: 422147), which were listed as likely pathogenic. This variant was not screened in the parents’ DNA  unrelated patients with rare genetic diseases together with analysis of biological processes to explore the implementation of whole-exome sequencing for diagnosis of rare genetic diseases in Bangladeshi children. Bangladesh is a small and densely populated developing country with limited healthcare resources. As a result, there is limited availability of targeted genetic tests for diagnosing a broad range of genetic disorders. WES provides a comprehensive front-line genomic test to efficiently diagnosis a broad range of rare genetic disorders. The five patients described here had complex phenotypic manifestations. Therefore, it was difficult to do targeted genetic testing with diagnosis-specific customized panels. In all five (5)patients, the mutations described are very likely to be etiologically related to the clinical pictures described.DHH that causes Proline to Arginine change at position 288 of Desert Hedgehog protein. DHH gene consists of 3 exons and is located on chromosome band 12q13.1232. It encodes a Desert Hedgehog signaling molecule that consists of 2 domains: a soluble amino-terminal “Hedge” domain (DHh-N: 23-198 amino acid) and a carboxyl-terminal “Hog” domain (DHh-C: 199 to 396 amino acid)33. The Hedgehog pathway plays a critical role in developmental processes, embryogenesis, cell proliferation and tissue regeneration36. DHH is important in gonadal differentiation39 as it is produced by Sertoli cells and is important for fetal Leydig cell differentiation40. Once cleaved, DHh-N domain functions as a signaling molecule while the DHh-C participates in autoprocessing regulation34. Disruption of the DHh auto-processing due to clinically relevant variants is associated with 46,XY gonadal dysgenesis (GD) or 46,XY sex reversal 741. We speculate that the p.Pro288Arg variant diminishes autoproteolysis activity of the DHh-C domain of DHH protein that lead to the clinical condition 46,XY gonadal dysgenesis (GD) or 46,XY sex reversal 7 in our patient  phenotype in the probands. They concluded that the presence of a homozygous 3503_3504delTC variant is sufficient to produce MLII44. Tappino et al. reported that its allele frequency is 51.1% (47/92) in their MLI and MLIII alpha/beta patients from diverse ethnic backgrounds 16. In addition, this variant was found worldwide and resulted most probably from a unique founder molecular lesion44. Functional studies demonstrate that the variant produces a truncated protein that is retained in the endoplasmic reticulum and not transported to the Golgi, and therefore does not form a mature subunit17. This variant has also been shown to result in absent or significantly decreased enzyme activity in patient fibroblasts46.Patient 2 presented with respiratory distress, congenital heart disease, craniofacial dysmorphology, and joint contractures Table . A compoGNPTAB that causes Methionine to Isoleucine change at position 991 of protein and leads to a premature termination codon 25 amino acids downstream. We hypothesize that this novel truncating mutation, p.Met991Ilefs*, along with another truncating frameshift mutation, p.Leu1168Glnfs*, is sufficient to result in MLII. A single cytosine insertion at codon 1,049 has been identified in a homozygous state in a male ML II patient resulting in a frameshift and a premature termination (p.Gly1049Argfs*16)47, and the insertion of the homologous mutation in the murine Gnptab results in mice displaying the clinical and biochemical features of ML II disease including the complete loss of GlcNAc-1-phosphotransferase activity48. Mucolipidosis II-related pathogenic variants, p.Gly1049Argfs* and p.Leu1168Glnfs*, inhibit the exit from the endoplasmic reticulum and proteolytic cleavage of GlcNAc-1-phosphotransferase precursor protein (GNPTAB)17. Almost all patients with classical severe MLII have nonsense or frameshift mutations in GNPTAB51. From the patient’s genetic data, clinical conditions, and the above-mentioned review of literature, we confirm that patient 2 is likely to be affected by Mucolipidosis II alpha/beta (MLII) . Mutations in BBS1 gene are the most common cause of BBS, accounting for about one-quarter of all cases. The most common pathogenic sequence alterations in BBS1 is the missense mutation p.M390R53 and is found in about 20–30% of BBS cases55. Schmid et al. found a homozygous variant c.479 G > A in the last nucleotide (splice site mutation) of exon 5 of BBS1 that causes aberrant splicing of BBS1 transcripts and co-segregated with the phenotype of retinitis pigmentosa56. Although, the clinical presentation of this patient is consistent with Bardet–Biedl Syndrome (BBS), given the vast number of loci associated with BBS (BBS1-BBS21) combined with the fact that no customized BBS targeted gene panel exists in Bangladesh, a decision was therefore taken to undertake WES. From the WES data, we analyzed all causal genes for BBS, but did not find any clinically relevant variants in those genes except for the c.1339 G > A variant in BBS1. Patient 3 meets the major clinical features of BBS with regard to hypothyroidism, polydactyly, obesity, strabismus, impaired intelligence in comparison to peers, and language delay. Therefore, we define the variant as likely pathogenic (criteria described in patient 1) and assume that it is associated with BBS  (Table SURF1 gene encodes a 300 amino acid long protein that contains two predicted transmembrane domains and acts as an important factor in cytochrome c oxidase (COX) assembly and maintenance. COX is the terminal component of the mitochondrial respiratory chain that mediates the transfer of electrons to oxygen58. In multiple literatures, it was established that missense pathogenic variants in SURF1is associated with deficient cytochromecoxidase assembly in Leigh syndrome patients60. SURF1-Associated Leigh Syndrome occurs due to null mutations accounts for 86% and missense mutations accounted for 14% of the disease alleles61. We hypothesize that this missense variant will produce an unstable surf1 protein that will be readily degraded. In addition, p.Gly77Arg missense variant is in trans configuration with another definitive deleterious variant, p.Arg264Serfs*. Teraoka et al. identified a missense variant c.820 T > G along with the deleterious variant p.Arg264Serfs* in a Japanese patient with Leigh syndrome (256000). Most of the clinical conditions of this patient are consistent with the clinical symptoms present in our patient (patient-4)25. CT Scan of the patient’s brain found bilateral cerebral and cerebellum leukodystrophy or ischemic change. A similar pattern of leukodystrophy was also found in a child carrying a homozygous variant, p.Arg264Serfs*, in SURF1. Most of the clinical conditions of this patient are also consistent with the clinical symptoms present in our patient (Patient-4)62. From the patient’s genotype (Patient-4), phenotype, and the above-mentioned review of literature, we hypothesize that the compound heterozygous variant p.Gly77Argand p.Arg264Serfs* are associated with this patient’s phenotype  complex28. AP-complex is ubiquitously expressed in neurons throughout the embryologic and postnatal developmental stages and interacts with delta2 and α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) glutamate receptors to selectively transport them from the trans-Golgi network to the postsynaptic somatodendritic domain63. The vast majority of pathogenic variants identified in this gene are truncating variants, such as nonsense variants, frameshifts, and canonical splice sites which often disrupt gene function by leading to a complete absence of the transcript through nonsense-mediated decay64. These mutations result in a non-functional AP-4 complex that leads to abnormal brain growth and development, which are associated with spastic paraplegia-47 (SPG47), intellectual disability, or AP-4 deficiency syndrome31.Patient 5 presented with tonic-clonic seizures in the context of a 48 h fever. On WES, a homozygous nonsense variant c.1216 C > Tin the exon 8 of AP4B1 have been reported64 which includes the c.1216 C > T variant found in our patient. This variant was reported in two unrelated AP4B1-associated SPG47 patients as compound heterozygotes31. Both were female and the disease was diagnosed at the age of 29 months and 36 months, respectively. The two patients had speech delay, infantile hypotonia, delayed motor development, developmental delay/intellectual disability, seizures, and lower extremity spasticity, but did not present with microcephaly. Our patient is clinically similar with regard to the seizures, absence of microcephaly, delayed speech and motor development. Spastic paraplegia was not seen in this patient, as this typically develops later in the course of the disease64. As the patient’s phenotype matches with other patients described in the literature, we hypothesize that the homozygous mutation, c.1216 C > T, is associated with Spastic Paraplegia-47  pathogenic variants in 65. We appreciate that genetic testing may not directly impact the course of the disorder, which, for neurodevelopmental disorders at least, is often chronic and without disorder-modifying treatments. However, there are a number of ways in which genetic testing can impact care pathways, which is true in Bangladesh to the same extent as other countries. Specifically, families generally seek genetic testing as it provides them with a sense of understanding regarding the origin of their child’s difficulties. It also avoids the child going though unnecessary diagnostic investigations (diagnostic odyssey). It also opens up the opportunity of expert counseling for the family regarding prognosis and other possible medical complications, which also offers to possibility of screening and early intervention. We, therefore, believe that even in a country with limited health resources there are many benefits to diagnostic testing.Our findings demonstrate the potential utility of WES as a first-line diagnostic approach in patients lacking a clear differential diagnosis in a developing country with limited healthcare resources. This approach will facilitate the identification of rare genetic diseases, accelerate rare disease research and ultimately help implement precision medical care4. The remaining patients were suspected of rare developmental disorders and were referred for WES. In each patient, the treating medical team noted a range of symptomatic manifestations, across different systems. The physicians collected phenotypic details, as summarized in Table From March 2019 to February 2020, 280 unrelated patients with neurodevelopmental disorders (NDDs), cancer, and rare genomic diseases were referred to NeuroGen Children’s Healthcare from multiple tertiary hospitals within the country. Of the 280 cases, 52 patients were referred for targeted cancer panel sequencing by their physicianThe study protocol was reviewed and approved by the Ethical Review Committee of the Faculty of Biological Sciences, University of Dhaka, and Institutional Review Board of Holy Family Red Crescent Medical College. Before participant enrollment, written informed consent including the use of peripheral blood and clinical data for research use and publication were obtained from the parents.Peripheral blood samples were collected from participants in an EDTA vacutainer tube. Genomic DNA was isolated using ReliaPrep™ Blood gDNA isolation kit  according to manufacturer instructions. The concentration and quality of DNA was determined using NanoPhotometer C40  67 for converting raw sequence reads to Binary Alignment Map (BAM) and Variant Call Format (VCF) v4.1 files. The DNA sequence was assembled and aligned to reference gene sequences based on the human genome build GRCH38/UCSC hg38. ANNOVAR (2018Apr16 version) was used for functional annotation of the variants. For genomic annotations, GenomeArc (http://genomearc.com/), a custom clinical genetic annotation tool, was used to annotate gnomAD variant frequencies, clinvar pathogenic mutations, and pLI. Variant classification analysis was conducted based on the American College of Medical Genetics (ACMG) guidelines13 and Sanger sequencing was used to confirm the variants68.Whole exome sequencing (WES) was conducted using the HiSeq sequencing platform. DNA libraries were prepared using the SureSelect XT library preparation kit and the SureSelect V7-Post Target Enrichment Kit. HiSeq uses a Burrows–Wheeler Aligner (BWA)® SV Gel and PCR Clean-Up System  according to the manufacturer’s instructions. Cycle sequencing was performed using purified PCR products as templates and BigDye® Terminator v3.1 . Bidirectional Sanger sequencing was performed using 3500 DNA Analyzer  to determine the sequence. Sanger sequencing data were analyzed using Sequence Scanner v2.0 .The recessive variants identified from WES were validated by Sanger sequencing using standard protocols. Validation primers (Supplementary Table Further information on research design is available in the Supplementary InformationReporting Summary


Patients in the intensive care unit (ICU) are known to be at increased risk of developing delirium, but the risk of subsequent neuropsychiatric disorders is unclear. We therefore sought to examine the association between the presence of delirium in the ICU and incident neuropsychiatric disorders  post-ICU stay among adult medical-surgical ICU patients.Retrospective cohort study utilizing clinical and administrative data from both inpatient and outpatient healthcare visits to identify the ICU cohort and diagnostic information 5 years prior to and 1 year post-ICU stay. Patients ≥ 18 years of age admitted to one of 14 medical-surgical ICUs across Alberta, Canada, January 1, 2014–June 30, 2016, and survived to hospital discharge were included. The main outcome of interest was a new diagnosis of any neuropsychiatric disorder 1 year post-ICU stay. The exposure variable was delirium during the ICU stay identified through any positive delirium screen by the Intensive Care Unit Delirium Screening Checklist (ICDSC) during the ICU stay.Of 16,005 unique patients with at least one ICU admission, 4033 patients were included in the study of which 1792 (44%) experienced delirium during their ICU stay. The overall cumulative incidence of any neuropsychiatric disorder during the subsequent year was 19.7% for ICU patients. After adjusting for hospital characteristics using log-binomial regression, patients with delirium during the ICU stay had a risk ratio (RR) of 1.14  of developing any neuropsychiatric disorder within 1 year post-ICU compared to those who did not experience delirium. Delirium was significantly associated with neurocognitive disorders , but not depressive disorders , anxiety , and trauma-and-stressor-related  disorders.The diagnosis of new onset of neurocognitive disorders is associated with ICU-acquired delirium. In this study, significant associations were not observed for depressive, anxiety, and trauma-and-stressor-related disorders. Delirium is an acute state characterized by an onset of confusion and decline in cognitive ability often occurring in hospitalized patients –5. The pRisk factors for delirium have similarly been implicated in the psychiatric morbidity of critically ill patients post-stay , 15. A nThe aim of the current study was to evaluate the association between delirium in the ICU and a new diagnosis of any neuropsychiatric disorder in adult medical-surgical ICU patients during 1 year post-ICU stay. We hypothesized that patients with delirium in the ICU would be at an increased risk of developing neuropsychiatric disorders during the 1-year follow-up period.This retrospective cohort study was conducted and reported in accordance with recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement . The ConWe identified all adult ICU patients (≥ 18 years) who were admitted to one of the 14 medical-surgical ICU across Alberta, Canada (population 4.2 million people), between January 1, 2014, and June 30, 2016 . Data waCanadian Enhancement of International Statistical Classification of Diseases, 10th Revision, fields for diagnostic codes. The NACRS captures data for all hospital-based and community-based emergency or ambulatory care and has up to 10 Canadian Enhancement of International Statistical Classification of Diseases, 10th Revision, fields for diagnostic codes. Physician Claims captures patients who received care from an outpatient clinic  and has up to 3 Canadian Enhancement of International Statistical Classification of Diseases, 9th Revision, fields for diagnostic codes. Vital Statistics data is provided to Alberta Health Services from Alberta Health. All deaths occurring in Alberta must be registered with Alberta Vital Statistics. The DAD, NACRS, and Physician Claims were available until October 31, 2017, and Vital Statistics were available until December 31, 2017, at the time of data extraction.We used clinical and administrative databases from Alberta Health Services and Alberta Health , previously successfully used in research studies , 25. eCrThe primary exposure was ever having delirium at any point during the ICU stay. Delirium was measured using the validated Intensive Care Delirium Screening Checklist (ICDSC) , which iany of the four neuropsychiatric disorders and then for each disorder separately.Neuropsychiatric disorders identified in previous systematic reviews to potentially be associated with delirium were included as outcome measures –33, incl. Characteristics included age, sex, ICU admission reason , Acute Physiology and Chronic Health Evaluation (APACHE) II score, Charlson Comorbidity Index (CCI), Glasgow Coma Scale (GCS), transfer delay ≥ 24 h, invasive mechanical ventilation (yes/no), non-invasive mechanical ventilation (yes/no), continuous renal replacement therapy (yes/no), vasoactive medications (yes/no), ICU size (categorized as < 20 beds vs. ≥ 20 beds), teaching hospital (yes/no), ICU length of stay (categorized as < 7 days vs. ≥ 7 days), and last Sequential Organ Failure Assessment (SOFA) score.Characteristics potentially associated with delirium during the ICU stay and neuropsychiatric disorders at a 1-year follow-up were identified a priori , 38. ChaU test for continuous non-normally distributed variables and chi-squared tests for binary variables. The cumulative incidence of any and each neuropsychiatric disorder was calculated by identifying the number of new cases of the neuropsychiatric disorder within 1 year (the numerator) divided by the total number of patients at risk of developing the disorder (the denominator). The incidence density of any and each neuropsychiatric disorder was calculated by identifying the number of new cases of the neuropsychiatric disorder within 1 year (the numerator) divided by the total number of person years for patients at risk of developing the disorder (the denominator). For patients who did not survive to 1 year post-ICU stay, time from hospital discharge to death was taken as time at risk.Descriptive statistics for demographic characteristics and covariates were examined using median (interquartile range [IQR]) for continuous variables and frequencies (with percentages) for categorical variables. Group comparisons were made using the Mann-Whitney http://www.r-project.org/).Associations between delirium in the ICU and new neuropsychiatric disorders were assessed using log-binominal regression analyses (in which exponentiated regression coefficients represent risk ratios). Supplementary analyses were also conducted for each disorder separately  using log-binomial regression analyses. We adjusted all models for all covariates previously identified. Analyses were conducted in STATA version 14  and R, version 3.5.1 . Of those, 6442 patients had a healthcare visit within the previous 5 years that included a diagnosis of a neuropsychiatric disorder, leaving 4033 patients who satisfied the inclusion criteria . The median age of ICU patients in this study was 60 years (IQR 46–71 years), 68% (n = 2744) were male, the most common reason for an ICU admission was medical , and the median length of ICU stay was 4.1 days (IQR 2.3–7.6 days). The median Charlson Comorbidity Index was 1 (IQR 0–3), median SOFA score upon ICU admission was 6 (IQR 3–9), and median APACHE II score upon ICU admission was 18 (IQR 13–24).Patient and hospital characteristics of the primary study cohort are summarized in Table During the 1-year follow-up period, 794 patients  had a healthcare visit for a newly diagnosed neuropsychiatric disorder. The cumulative incidences of depressive, anxiety, trauma-and-stressor-related, and neurocognitive disorders were 10.1% (95% CI 9.2–11.1%), 8.8% (95% CI 8.0–9.7%), 2.9% (95% CI 2.3–3.4%), and 3.5% (95% CI 2.9–4.1%), respectively. The incidence density for those who developed any neuropsychiatric disorder during the 1-year follow-up was 213 (95% CI 200–226) per 1000 person years. The incidence densities of depressive, anxiety, trauma-and-stressor-related, and neurocognitive disorders were 110 (95% CI 100–120) per 1000 person years, 96 (95% CI 86–105) per 1000 person years, 31 (95% CI 25–36) per 1000 person years, and 38 (95% CI 32–44) per 1000 person years, respectively.Patients who ever had delirium in the ICU had 1.14 (95% CI 0.98–1.33) times the risk of developing a neuropsychiatric disorder within 1 year compared to those who never had delirium in the ICU Table . The adjThis retrospective cohort study examined the association between delirium in the ICU and subsequent healthcare visits for a neuropsychiatric disorder, captured in both inpatient and outpatient settings. There was a high prevalence of delirium in the study cohort, which is consistent with the existing literature on the prevalence of delirium in the ICU , 39. In There is a growing awareness of neuropsychiatric disorders following an ICU stay , 40. A sOur study showed a significant association between delirium in the ICU and a subsequent diagnosis of a neurocognitive disorder. However, our study did not find significant associations for depressive, anxiety, and trauma-and-stressor-related disorders, potentially due to the comorbid relationship between depression and anxiety, as depressive disorders may present more prominent symptoms leading to the misclassification of anxiety disorders as depressive disorders . FurtherStrengths of this study include the large sample size and population-based nature of the data, as well as the ability to control for a number of risk factors for delirium and neuropsychiatric disorders. Data were also available for pre-existing neuropsychiatric disorders. Moreover, this study was able to utilize a provincial, validated delirium identification tool . Our stuOur data suggest that patients who experience delirium during the ICU are at increased risk of developing a neurocognitive disorder in the 1 year following their ICU stay. This risk appears to be dose dependent. Significant associations with depressive, anxiety, and trauma-and-stressor-related disorders were not observed, but the confidence intervals associated with risk ratios for these disorders do not necessarily preclude the existence of clinically significant associations between delirium and these conditions. Future clinical and research efforts should focus on early detection and management of delirium to mitigate the potential development of neurocognitive disorders post-ICU stay. Consideration should be given to monitoring ICU patients with delirium for long-term neurocognitive disorders.Additional file 1. Frequency of Diagnostic Codes for Each Neuropsychiatric Disorder Per Visit Per Database for All Patient Data.: The data presented in additional file 1 present frequency of diagnostic codes for each neuropsychiatric disorder per visit per database  for all patient data.Additional file 2. Characteristics of Those With and Without Pre-Existing Neuropsychiatric Disorders. The data presented in additional file 2 compare characteristics of patients with and without pre-existing neuropsychiatric disorders from five years prior to ICU admission.Additional file 3. Unadjusted Risk Ratios for Delirium, Patient Characteristics and Neuropsychiatric Disorders. The data presented in additional file 3 present detailed unadjusted risk ratios for delirium, patient characteristics and each neuropsychiatric disorder.Additional file 4. Adjusted Models for Delirium and Neuropsychiatric Disorders. The data presented in additional file Additional file 5. Sensitivity Analyses Between Delirium and No Delirium/one calendar day/two or more calendar days of Delirium in the Intensive Care Unit. The data presented in additional file 5 is the outputs of a sensitivity analysis between delirium and no delirium/one calendar day/two or more calendar days of delirium in the ICU.


Circulating levels of cardiac troponin I (cTnI) after ST-segment elevation myocardial infarction (STEMI) were considered as prognostic factors for predicting the incidence of major adverse cardiovascular events (MACE). △cTnI is the difference between peak cTnI after primary percutaneous coronary intervention (PPCI) and cTnI on initial admission.  This study aimed to assess the relationship between △cTnI, the ratio of △cTnI to cTnI on initial admission, and the incidence of MACE during the follow-up period.  A total of 2596 patients with cTnI measured upon admission and one-time measurement of cTnI during hospitalization were enrolled. p < 0.001), higher GRACE score (p = 0.038), CK-MB (p = 0.023), and Myoglobin (p < 0.001). On the K–M survival curves, the incidence of MACE, mortality, and angina pectoris were significantly higher in the group with maximum △cTnI .  In the adjusted models of the survival receiver operating characteristic (ROC) curve, △cTnI and the ratio of △cTnI to cTnI on initial admission have stronger discrimination power of MACE (area under curve (AUC) 0.730 and 0.717) compared with peak cTnI after PPCI and cTnI at admission . Multivariate Cox regression analysis identified △cTnI (hazard ratio (HR) 1.018, 95% confidence interval (CI) 1.001 to 1.035) as a relevant factor for MACE during follow-up. △cTnI was divided into quartiles, and maximum △ cTnI between 4.845 and 19.073 ng/ml comprised more patients with anterior wall myocardial infarction ( The △cTnI level and the ratio of △cTnI have stronger discrimination power of predicting the incidence of MACE. The group with maximum △cTnI has higher incidence of MACE, mortality, and angina pectoris during the follow-up period. Cardiac troponin I (cTnI) is a highly specific and sensitive biomarker of cardiac injury and a regulatory protein with cytosolic and structural compartments within the cardiac myocytes . cTnI isFrom a total of 4064 patients who presented at Fuwai Hospital in Beijing, China, between January 2010 and July 2018, 3586 consecutive STEMI patients  were enrolled (478 patients who were lost to follow-up were excluded from the study). All patients were referred to the coronary catheterization center with the diagnosis of acute STEMI fulfilling the criteria for PPCI according to the guidelines [Patient records including demographics, medical history, physical examination, blood test results, electrocardiography (ECG), echocardiography data, and discharge medication regimen were reviewed. Blood testing was performed at the clinical laboratory in Fuwai Hospital. Blood samples for cTnI measurement were acquired on admission and after PPCI. Patients were required to have at least one measurement of the cTnI level during hospitalization. However, of the 3586 patients, those who did not have a valid cTnI result and those with missing postdischarge follow-up data were also excluded. Finally, 2598 patients were included in the analysis. The study flow chart is shown in 2). The no-reflow phenomenon was defined as thrombolysis in a myocardial infarction (TIMI) flow grade <3 after PPCI.In this study, △cTnI was defined as the value calculated as the post-PPCI peak cTnI value minus the first admission cTnI value. STEMI was defined as continuous chest pain lasting >30 min, an elevated troponin I level, and an ECG finding of ST-segment elevation >0.1 mV in at least two contiguous leads or a new left bundle-branch block on an 18-lead ECG . HyperteBlood samples were drawn from an antecubital vein in the morning after overnight fasting and collected into vacuum tubes containing EDTA for the measurement of plasma lipid and lipoprotein levels. TC, (HDL-C), LDL-C, TG, and homocysteine levels were analyzed by colorimetric enzymatic assays using an auto analyzer at the chemistry laboratory of the Fuwai Hospital Peking of Union Medical College. The level of C-reactive protein (CRP) was measured by biodirectional lateral flow immunoassay according to the procedure at the same chemistry laboratory.Myocardial biomarkers were measured as follows: cTnI levels were measured on blood samples collected upon admission and after PPCI and serum was separated internally. The Abbott ARCHITECTi2000SR  immunoassay system (batch number: 73099UI00) and Beckman UniCelDXI800  access immunoassay system (batch number: 624362) were used to analyze the cTnI level. The detection limit of the assay is 0.02 ng/ml, and the decision limit for the diagnosis of MI is 0.07 ng/ml.PPCI  was performed using standard criteria. Heparin or bivalirudin was used as periprocedural anticoagulant therapy. Glycoprotein IIb/IIIa inhibitors were used at the discretion of the operator. Commercially available stents were used. The dual antiplatelet therapy following PPCI consisted of oral aspirin (80–325 mg/day continued indefinitely) and a P2Y12 inhibitor for at least 12 months. Other medications were prescribed at the discretion of the trained attending physicians.All ECG tracings from the emergency response teams and emergency department were collected and analyzed. Additional 18-lead ECGs were obtained upon arrival to the emergency department and twice daily thereafter until discharge. Each participant was scanned by trained ultrasonographers using the color Doppler ultrasonic diagnostic apparatus, cardiac ultrasonic measurement software, and a linear array transducer at a frequency of 12 MHz.For adverse events that occurred at follow-up, the following were evaluated: overall mortality, recurrence of MI , and stroke.χ2 test for categorical variables. Univariable and multivariable Cox proportional hazards regression modeling was performed to characterize predictors of MACE. Categorical variables included the △cTnI group, target lesion types of culprit vessels of MI, status of target organ thrombosis, status of the target organ occlusion, and whether the target lesion involves branches. Continuous variables included the △cTnI level difference before and after PPCI and the GRACE score. Significant variables analyzed were reported with their respective hazard ratios and 95% confidence limits. All p values are two-tailed, and statistical significance was determined at p < 0.05. Time-dependent ROC curves were performed with R language version i386 3.6.2. The other analyses were performed with SPSS version 20.0 statistical software .The normal distribution of outcome variables was confirmed by Kolmogorov–Smirnov tests. Baseline parameters and major adverse events during follow-up were presented as median (interquartile range) for continuous variables and as frequency and percentage for categorical variables. The relationship between baseline clinical, ECG, angiography characteristics, follow-up outcomes, and peak levels was assessed by the Spearman correlation. Survival analysis was performed with the Kaplan–Meier method. To assess the discrimination utility of cTnI, we plotted the time-dependent receiver operating characteristic (ROC) curves conducted by R language in order to obviate the limitation of potentially biased due to censoring. The predictive values of the ratio of delta cTnI to cTnI measured prior PPCI, delta cTnI, peak cTnI level post-PPCI, and the first admission cTnI level were obtained in the range of 0-1 using the logistic regression model by controlling the following: history of hypertension, DM, hyperlipidemia, coronary artery bypass grafting, PCI, smoking, chest pain onset to hospital stay, Apo A, Apo B, CRP, TC, TG, HDL-C, LDL-C, and medication history . The ROC curves were obtained by incorporating three predictive values. We tabulated the baseline characteristics of the cohort and, then, examined the bivariate association between these variables and △cTnI quartiles. Differences across △cTnI quartiles were evaluated by the analysis of variance  or Kruskal–Wallis test (skewed variables) for continuous variables and with the p < 0.001).The median (interquartile range) time between symptom onsets to admission was 7 h (8 h). Patients' baseline clinical characteristics are summarized in p = 0.005), status of target organ thrombosis , status of target organ with complete occlusion , left atrial diameter (LAD) on discharge , LVEDV on discharge , and mortality on the median 2-year follow-up . Moreover, an independent significantly negative correlation was found between △cTnI and the time from symptom onset to admission , TIMI flow grade pre-PPCI , and LVEF on discharge .Correlation coefficients between cTnI and angiography characteristics, outcome at 2-year follow-up, and echocardiography measurement at discharge are shown in p < 0.001), higher Global Registry of Acute Coronary Events (GRACE) score (p = 0.038), creatine kinase MB (p = 0.023), and myohemoglobin (p < 0.001), while age, sex, the no-reflow phenomenon, triple-vessel disease, the number of stents, and risk factors including hypertension, hyperlipidemia, and DM failed to present statistical difference between groups (p = 0.042), Q2 group , and uric acid  as relevant factors for MACE during follow-up (The characteristics of the group with the maximum difference (fourth group) in cTnI levels (ng/mL) that ranged from 19.161 to 101.66  at baseline, as shown in n groups . Multivaollow-up .p = 0.035, 0.049, 0.015, 0.026) (The median follow-up time was 2 (range <1–8.35) years. The Kaplan–Meier curves depicted a cumulative probability of MACE, mortality, recurrent myocardial infarction, and angina pectoris for patients stratified into quartiles of the △cTnI level at enrollment  .This study investigated defined variables in a large-scale, well-described, and contemporary-treated STEMI population from China. The novelty of the study was the assessment of the discrimination value of the △cTnI and the ratio of △cTnI to cTnI on initial admission to the incidence of MACE which remains largely unknown. The researchers followed strict inclusion and exclusion criteria, which facilitated a reasonably streamlined and comparable hospital flow for all subjects. On the other hand, the use of time-dependent ROC curves may obviate potentially biased due to censoring.p ≤ 0.001), minimum vessel diameter prior PPCI , LAD on discharge , LVEDV on discharge , and LVEF on discharge .Several prior investigations supported that the early release of cTnI following AMI is caused by the washout mechanism from the infarcted zone into the serum, a process which is facilitated by early restoration of blood flow to the infarcted tissue –13. In tp = 0.002), complete occlusion of the target lesion (p ≤ 0.001), TIMI blood flow grade 0 at pre-PPCI (p ≤ 0.001), and statistically significant lower use of cardiovascular-related drugs at post-PPCI than other groups. Based on the Kaplan–Meier analysis of the probability of death, the mortality rate of the group with the maximum △cTnI (median 52.00) was significantly higher than that of other groups.Many studies reported –22 that p = 0.02) and severe heart failure  at 3 years in patients with anterior STEMI who underwent PPCI. These observations, together with the present results, tend to support the characteristics of the fourth group of the △cTnI level with the maximum cases of anterior wall MI and higher risk for MACE. The pathophysiology of the no-reflow phenomenon is complex, and a series of consistent data has [p = 0.01), cerebrovascular accident , postprocedure bleeding , and cardiogenic shock . The no-reflow phenomenon is a process in which prolonged ischemia caused changes in endothelial cells, and optimal treatment of hyperglycemia is a significant target in preventing [p = 0.337). This was possibly caused by the number of participants and the rare occurrence of the no-reflow phenomenon after PPCI in our study.Sezer et al.  reporteddata has , 28 cleadata has  that theeventing , 31. On p = 0.272) and triple-vessel disease (p = 0.984) have a significant statistical difference with other groups. Because of the inadequate calibration, the actual prevalence of the four groups according to the △cTnI level fluctuation may need to be viewed with caution in Chinese population with STEMI following PPCI. Several studies have reported the predictive accuracy of the GRACE risk score in different patient populations. A study of contemporary populations [p ≤ 0.001). Furthermore, mathematical models and artificial intelligence have recently come in help in the setting, and integration into the clinical workflow might significantly improve patient outcomes [The findings from studies , 33 emphulations  with a vulations  concludeoutcomes . The novNevertheless, this study has several potential limitations. Firstly, it is a single-center, retrospective study design with strict inclusion criteria, and many patients who did not have a valid cTnI result were excluded, which resulted in selection bias. Secondly, patients have been enrolled during a long span of time, which could bring about a confounding effect due to the improvements of interventional techniques and progress in medication. Multicenter studies with a larger sample of patients admitted in a short period would be preferred to validate the results of this study. While the discrepancies in discharge medication were explored, a potentially unbalanced distribution of missing values may have influenced the results. Finally, we did not analyze some acute phase biomarkers such as ST-segment recovery and reperfusion ventricular arrhythmia “bursts” which are also related to the outcome in our models. Thus, the relative predictive information provided by △cTnI simultaneously considered with such additional biomarkers remains an important area for future research.p < 0.001), a higher GRACE score (p = 0.038), myohemoglobin (p < 0.001), and TIMI flow grade 0 at pre-PPCI (p < 0.001).The main findings of this study are as follows: (1) △cTnI and the ratio of △cTnI to cTnI on initial admission were significant prognostic indicators in patients with MACE compared with first cTnI and peak cTnI after PPCI. (2) The maximum △cTnI level (median 52.00) was associated with a higher incidence of MACE, mortality, and angina pectoris at media 2-year follow-up than the other groups. (3) A higher number of patients with the maximum △cTnI level more likely had anterior wall MI (Our findings supported that repeated measurements of cTnI and △cTnI could provide significant incremental information for risk stratification of STEMI patients who underwent PPCI. It represents a valuable, inexpensive, and readily available tool for assessment of risk, especially of long-term MACE; in addition, our results suggest that routine use of △cTnI values provide complementary information to well-established clinical risk factors. Furthermore, one can envision a △cTnI biomarker-guided approach to implement aggressive medical treatment strategies before discharge in patients at increased risk. Finally, it could provide help in the future selection of individuals considered for inclusion in clinical trials aimed at improving outcomes in such a population.


LPA gene SNPs rs10455872 and rs3798220 are associated with the incident development of microvascular complications in type 2 diabetes.Microvascular disease in type 2 diabetes is a significant cause of end-stage renal disease, blindness and peripheral neuropathy. The strict control of known risk factors, e.g. lifestyle, hyperglycaemia, hypertension and dyslipidaemia, reduces the incidence of microvascular complications, but a residual risk remains. Lipoprotein (a) [Lp(a)] is a strong risk factor for macrovascular disease in the general population. We hypothesised that plasma Lp(a) levels and the n = 1886 individuals with type 2 diabetes, mean follow-up time = 6.97 years). To assess the relationship between plasma Lp(a) levels and the LPA SNPs with each newly developed microvascular complication , Cox proportional hazards models were applied and adjusted for risk factors for microvascular complications .Analyses were performed of data from the DiaGene study, a prospective study for complications of type 2 diabetes, collected in the city of Eindhoven, the Netherlands  levels.No significant associations of Lp(a) plasma levels and the LPA SNPs with known effect on Lp(a) plasma levels with the development of microvascular complications in type 2 diabetes. This indicates that Lp(a) does not play a major role in the development of microvascular complications. However, larger studies are needed to exclude minimal effects of Lp(a) on the development of microvascular complications.Our data show no association between Lp(a) plasma levels and the The online version of this article (10.1007/s00125-020-05120-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Microvascular complications greatly reduce the quality of life of individuals with type 2 diabetes. Microvascular damage causes end-stage renal disease, blindness and peripheral neuropathy –3. StricLPA SNPs, rs10455872 and rs3798220 influence Lp(a) plasma levels, and are associated with a lower number of Lp(a) KIV repeats /eGFRbaseline) × 100. Model 1 was adjusted for sex and age. Model 2 was adjusted for the following additional risk factors for microvascular complications: MAP, non-HDL-cholesterol, HDL-cholesterol, HbA1c, BMI, duration of type 2 diabetes and smoking. In order to detect whether there was any other form of relationship we may have missed by analysing Lp(a) linearly or in quartiles, we performed non-linear spline analyses. More specifically, we applied P-splines  in both the logistic regression as well as Cox models [LPA SNPs carrier status associates with Lp(a) plasma concentration, one-way ANOVA was applied. We tested the Hardy–Weinberg equilibrium before incorporating the LPA SNPs in the analyses. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS 25.0 . Non-linear spline analyses were performed using R version 3.6.0 (https://www.r-project.org/), including packages ‘mgcv’ and ‘survival’. For cross-sectional and prospective analyses concerning Lp(a) as binary predictor, we had 80% power to detect an OR of 1.4 and an HR of 1.5 for each microvascular endpoint, respectively. And for cross-sectional and prospective analyses with Lp(a) as continuous predictor, we had 80% power to detect an OR of 1.09 and an HR of 1.013 for each microvascular endpoint, respectively. Moreover, for genetic analyses, we had 80% power to detect an HR of 1.7 for each separate SNP (rs10455872 and rs3798220) separately. ESM Table To compare baseline variables, the independent samples 30 mg/dl . However30 mg/dl . In addi30 mg/dl , we perfx models . This inp < 0.001), had a later age of onset of type 2 diabetes , a longer duration of type 2 diabetes  and higher MAP  than those who were did not develop microvascular complications.The baseline characteristics and the number (and percentage) of individuals with data missing per characteristic of the study population are shown in Table p = 0.71). Additional analyses with Lp(a) as a continuous variable or divided into quartiles did not change these results .In cross-sectional analyses at baseline (ESM Table p = 0.35). Additional analyses with Lp(a) as a continuous variable or divided into quartiles did not change these results .Table LPA SNPs rs10455872 and rs3798220 were in Hardy–Weinberg equilibrium , respectively. Minor allele frequencies of rs10455872 and rs3798220 were 0.0648 and 0.0156, respectively (see details on genotype distribution and mean Lp(a) plasma concentration according to the carrier status of the LPA SNPs in Table For the entire study population independent of microvascular complications at baseline the LPA SNPs, according to the additive genetic model. For both LPA SNPs, carriers of the minor allele did not have significant HRs for any of the microvascular complications. These results did not change when the separate microvascular endpoints were taken together as one composite endpoint (data not shown). Furthermore, these results did not change when analysing the two SNPs together as a genetic risk score  plasma levels and LPA SNPs rs10455872 and rs3798220, which, notably, did have their known significant effects on Lp(a) plasma levels. Moreover, in our DiaGene cohort the two SNPs rs10455872 and rs3798220 explained 20% and 8% of the variation in Lp(a) levels, respectively, and jointly explained 30% of the variance. This is relatively high compared with other type 2 diabetes populations , where rs10455872 explained approximately 3% of the Lp(a) variation [In the general population, Lp(a) is a causal risk factor for macrovascular complications . The risariation . Howeverariation . OverallThe two prospective analyses in a South Korean population by Yun et al pointed at a positive association between Lp(a) plasma levels and both diabetic retinopathy and nephropathy , 23. OneSample storage and method used to determine Lp(a) plasma concentration both influence measurements , 47. SevIn our type 2 diabetes population, Lp(a) levels were relatively low compared with reports in the general population . IndividLPA SNP rs10455872 is strongly associated with the KIV size and showed no association with our microvascular endpoints [Finally, the KIV size remains an essential property of Lp(a). The KIV size is independently inversely associated with macrovascular disease in the general population . Likewisndpoints . HoweverLPA SNPs. Moreover, we used an immunoassay with the least Lp(a) concentration-dependent bias of the available immunoassays, and this immunoassay has a correlation coefficient (r) of 0.99 with values determined by the ‘gold standard’ for measuring Lp(a) (an ELISA method) [1c, as a known continuous risk factor for microvascular disease, in the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) study resulted in an HR of 1.14  for microvascular disease after 5 years of follow-up [Strengths of our study are the prospective study design, relatively large type 2 diabetes cohort, the meticulous collection of phenotypic and risk factor data, the investigation of all microvascular endpoints and the concomitant investigation of the effects of  method) , 47. Furollow-up . This coIn conclusion, our data indicate that Lp(a) plasma levels do not play a major role in the development of microvascular complications in individuals from European descent with type 2 diabetes. Larger studies than ours may be able to detect very small effects of Lp(a) levels on these outcomes. Future studies taking the Lp(a) KIV size into account can further elucidate whether or not any other aspect of the Lp(a) particle is associated with the microvascular complications of type 2 diabetes.ESM(PDF 278 kb)


Face masks have been employed in the COVID-19 pandemic plans as a public and personal health control measure against the spread of SARS-CoV-2. In Poland, obligatory wearing of masks in public spaces was introduced on April 10th, 2020; a relaxation of previous universal measures was announced on May 29th, 2020, limiting use to indoor public places.To assess use of masks or other protective devices in public spaces in Poland during the SARS-Cov-2 epidemic.n = 2460 observations), using a structured checklist.A non-participatory covert observational study was conducted on three dates, (10.05/18.05/25.05.2020) at public spaces in 13 regions with different risks. Ten consecutive individuals were observed by each of 82 medical students (n = 552/750) were using masks on date 1, 66.5% (544/818) on date 2; and 65.7% (516/785) on date 3. Cloth masks predominated on all dates (64.7–62.3%-62.6%), followed by medical (23.4–28.5%-26.9%). Being female (OR = 1.77–1.47-1.53 respectively) and location in a closed space (OR = 2.60–2.59-2.32) were each associated with higher usage. Participants in sports were about two times less likely to use masks (OR = 0.64–0.53-0.53) as compared to other activities. The proportion using masks correctly decreased gradually over time . More females wore masks correctly . Uncovered noses (47.3–52.7%) and masks around the neck (39.2–42.6%) were the most frequent incorrect practices.Among 2353 observed persons, the female/male ratios were 1.0, 1.1, and 1.0 on the three dates. Almost three quarters - 73.6% (Practices were not in line with official recommendations, especially among males, and deteriorated over time. Cloth masks were predominantly used in public spaces. Health promotion, through utilizing all available communication channels, would be helpful to increase compliance. The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) pandemic that begun in the Chinese province of Hubei quickly became a global threat . As of DStrategies to transition out of lockdown , 6, carrEither disposable surgical masks or reusable cotton ones are protective. Wearing masks in public places is seen as an important contribution to containing viral spread, especially when physical distancing is not possible or unpredictable . LocatioIn the beginning of the pandemic, mask-wearing was controversial among leading health organizations, such as the World Health Organization (WHO) . HoweverThe European Centre for Disease Control (ECDC) recommends use in the community, especially when visiting busy, closed spaces . Formal Different regulations have been in place in central Europe since the epidemic began, including Poland, Slovakia and Czechia , 18. In h, 2020, 6 weeks after the country’s first laboratory confirmed SARS-Cov-2 case had been reported, a new control measure was announced [In Poland, obligatory mask wearing was not introduced in the initial phase of the pandemic. On April 10nnounced . It becannounced . A relaxnnounced . The oblReports on use of masks by the general public as a preventive measure to limit transmission are scant, especially in European countries. Therefore, the objective of this study was to assess use of masks or other protective devices in public spaces in Poland during the epidemic and to evaluate influencing determinants.In Poland, mandatory face covering in public spaces was introduced in the beginning of April, 2020 . In the According to the latest census, Poland has a population of 38,383,000 . The repAt 2 pm, at each time point described above, eighty two 4th year medical students observed ten consecutive individuals of > 4 years of age while venturing out into public spaces (820 observations per time point) which were divided into two categories: open/enclosed space. The age frame > 4 years was arbitrarily chosen due to the governmental regulation requiring mask use for children older than 4 years and adults . Two typField notes were taken at observation points. Next, data were entered to MS Excel 2013. Data quality was assured by the review of data completeness by the research team (OP and MG). Data were validated using STATISTICA PL Version 12.5 . Our main outcome was mask usage. For socio-demographic variables, gender was coded as one for males, and two for females. A place of residence variable was arbitrarily divided into 2 categories: a city up to 50,000 citizens and > 50,000 citizens; a region regarding pandemic risk – into: high-risk (R > 1) and low-risk/very low-risk areas (R ≤ 1.0); a type of activity observed – into playing sports/other activities. A bivariate analysis assessed the demographic characteristics , together with the participant location during the observation (open/closed space), and the type of activity observed, associated with the outcome variable. The categorical variable groups were compared using the chi-square test with Yates correction and Fisher’s exact test. For observations made at each of the 3 time points, standard single-outcome logistic regression models were built for the predicted outcome variable (mask usage); all models were then reduced by the use of a stepwise selection and a backward procedure  with theThe general characteristics of study participants by each time point is presented in Table The percentage of those using masks was 73.6% (552/750) at the first time point, decreasing a week later  and then – 2 weeks later . Table p < 0.01 for the three time points). A decreasing trend was observed in relation to mask use at three time points for both: the general public and females. In the case of males, a decrease in usage was seen during the second observation (68.1% vs 58.9%), however, the percentage of those using masks increased slightly (59.9%) a week later.Significantly more females than males used masks (p < 0.01) was observed between masks use in the open/enclosed locations at the three time points; Table Masks usage in both - enclosed and open space decreased among study participants during the 3-week observation . A statistically significant difference (p ≤ 0.01). The decrease of mask usage over time was more evident among those in sports than among the other activities.The percentage of people wearing masks at three time points in relation to the activity performed is presented at Table p = 0.004). There were no statistically significant differences observed in masks usage at any of three time points (p > 0.05) regarding the place of residence.As reported in Table p = 0.002; p = 0.03; p = 0.01 respectively). Participants observed in the closed spaces were more likely to use masks than those outdoors . Interestingly, participants in sports were about two times less likely to use masks as compared to other activities. .Multiple logistic regression analysis showed that participant gender, location during the observation and type of activity were independent predictors for mask usage Table . Femalesp = 0.045; p = 0.83; p = 0.0008 respectively). The differences in the correct manner of masks use were not significant regarding location and activity (p > 0.05).The proportion using masks correctly decreased gradually over time . A statistically significant between-gender difference was noted in the 1st and 3rd week of observation, with more females wearing masks correctly ; however, more participants used N95 respirators correctly when compared to other types of masks (p = 0.0008) at the 3rd time point. At all 3 time points uncovered noses  and masks around the neck  were the most common incorrect practices, followed by touching masks by hands  and incorrect mask fixation .The correct manner of mask use by type and time point is presented in Table Overall, compliance with wearing masks by the general public in Poland was good in the beginning of the study period (74%), however, it decreased (to 66%) in the 2nd and 3rd week of the observation, with a predominance of cloth masks observed. Female gender and closed space were each associated with higher mask usage. Those playing sports had about two times lower chance to use masks. The percentage of those using masks in the correct manner also decreased gradually over time, mainly due to a decreasing trend observed for medical and cloth masks; significantly more females correctly used masks. Breaches in nose covering and hanging masks around the neck were the most common incorrect practices while wearing a mask.The willingness of the general public plays a decisive role in achieving the successful implementation of protective measures . HoweverThe study found that in May 2020, when wearing masks in public spaces was compulsory in Poland, face covering was relatively high (74%). According to Ipsos , in the Concerns about the pandemic, combined with the perceived risk of infection may explain the high rates of masks use in our study. The strong association between respondents’ risk perceptions and taking comprehensive precautionary measures against influenza, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) infections has been reported previously –32. ApprOther studies found extreme variation in COVID-19-related face coverings worldwide , 34. As The concept of collective responsibility versus personal freedom has been important in the context of face masks usage since the beginning of this pandemic. According to Gostin , compulsThe decreasing trend regarding mask usage by the general public in Poland through the 3 weeks of observation, which illustrates behavioral change, may be due to a gradual decrease in concerns about the pandemic over time and lowering in the perceived SARS-Cov-2 infection risk. Less strict supervision, monitoring and inadequate governmental regulation enforcement by the relevant services may also influence the rapid decrease in compliance rates. Although failure to comply with the government regulation could result in a fine of up to Polish zloty (PLN) 500 to PLN 30,000, according to the National Police Headquarters, between April 15th and May 31st 2020, 40,000 citizens were instructed on correct mask use; only around 13,000 were fined for not using a mask in the public space, around 5000 applications were made to the court to fine individuals for not wearing a mask .Numerous examples of political leaders, including the Polish president and the prime minister not using masks in public spaces, which was transparently reported by the media –41, coulVarious factors, including socio-demographic characteristics, social context and individual values can affect the subjects’ perception of their actual risk of disease, as well as influence their worries about a pandemic , 42, 43.Although not assessed in this study, younger respondents are reported as less likely to use masks in order to mitigate the chances of spreading and contracting SARS-COV-2 ; this miWearing masks when out in public was significantly lower in open spaces during the three-week observation, and decreased to a greater extent (10%) over time, when compared to enclosed spaces. During the study period government officials announced that in the upcoming weeks it would not be necessary to wear masks while walking on the street, in parks or during sport activities outdoors, e.g. riding a bike ; this coTo control the infection source, as well as to self-protect, cloth masks, as recommended by the WHO, CDC, and ECDC , 14, 16 Notably, compliance of correct mask use was poor – only two thirds of those wearing masks  wore them correctly at the first time point, this decreased over time, with 55% wearing cloth masks and 60% - medical ones. Although N95 respiratory masks were rarely used by the public, compliance with correct use was the best and increased over time. One of the possible interpretations of this observation could be that the potential N95 respiratory masks wearers were more health oriented and health educated. People with high health literacy skills are more competent in related to public health outbreak controls , 44, 49.At all 3 time points breaches in nose covering and hanging masks around the neck were the most common incorrect practices while wearing a mask. Such practices elevate infection risk regarding SARS-Cov-2 and other airborne respiratory pathogens. Notably, the pathogens may settle on the surface of used masks layers, resulting in mask contamination  which inTo our knowledge, this is the first study to investigate masks use in the general population in Poland, as well as in the European Union (EU). The mask usage has been assessed by a non-participatory covert observational study. Other methods to assess the uptake of facemask against respiratory infections, include observational studies (most of them standard interviewer-administered surveys) and cases series  . NeverthOne particular strength of this study is that participants were observed during an actual continuing outbreak - data collection started 3 weeks after an introduction of the regulation about obligatory mask use in the public space. Furthermore, by covert observation, the Hawthorne effect has been limited as the behavior of participants was not altered . In addiThe results show an essential difference in how the general public responds to the SARS-Cov-2 pandemic in the context of mask usage. Practices were found to be inadequate, especially among males, and tended to decrease over time, more significantly among those wearing cloth masks than when compared to those wearing N95 respirators. This message can be used to target specific vulnerable groups when developing public health campaigns which should be then rigorously evaluated for their effectiveness. Among those using masks, every third individual wore them incorrectly. Therefore, awareness campaigns regarding the need for the proper usage of face masks - by utilizing all communication channels available - would be helpful during this pandemic . The resIn conclusion, this study identified topics that may need attitudinal modification to improve self-protection against SARS-Cov-2 infection and community spread of the virus. There is now an ideal moment to implement these adjustments.


Diminished ovarian reserve (DOR) is the weakening of ovarian oocyte production and quality. It will further become premature ovarian failure without timely cure. However, disease pathology and diagnostic markers are still incompletely understood. Liu-Wei-Di-Huang (LWDH) pill, a traditional Chinese medicine formula, is commonly used in the treatment of DOR in China. To explore the mechanism of the effect of LWDH on in vitro fertilization (IVF) outcomes in patients with DOR, a pseudotargeted metabolomics study combined with multivariate data processing strategy was carried out. A liquid chromatography tandem mass spectrometry-based metabolomics approach was applied to characterize metabolic biomarker candidates. Multiple pattern recognition was used to determine groups and confirm important variables. A total of 21 potential biomarkers were characterized, and related metabolic pathways were identified. The study displayed that the established pseudotargeted metabolomics strategy is a powerful approach for investigating the mechanism of DOR and LWDH. In addition, the approach may highlight biomarkers and metabolic pathways and can capture subtle metabolite changes from headache, which may lead to an improved mechanism understanding of DOR diseases and LWDH treatment. Diminished ovarian reserve (DOR) is often found in the process of long-term infertility seeking assisted pregnancy, which affects female sex hormone levels and fertility . PreviouFollicular fluid is a product of secretion of granulosa cells and testicular cells through the serum follicular barrier to provide nutrients for oocyte development . The chaHPLC-grade methanol, acetonitrile, water, ammonium acetate, and formic acid were all supplied by Fisher Scientific . The internal standards of chloramphenicol and clenbuterol were from Sigma-Aldrich . In our study, 3.2 kg of prepared radix rehmanniae, 1.6 kg of prepared rhizoma dioscoreae, 1.6 kg of prepared fructus corni, 1.2 kg of prepared tuckahoe, 1.2 kg of prepared rhizoma alismatis, and 1.2 kg of prepared tree peony bark were crushed by pulverizer. Then, the refine honey was added to the herbal powder at a ratio of 3 to 10. They were kneaded for half an hour and prepared into LWDH pills with a mold. LWDH pills were obtained from the Affiliated Hospital of Shandong University of Traditional Chinese Medicine  and authenticated by Dr. Sun .n = 84) were recruited, and their follicular fluid was collected at the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, from May 2018 to February 2019. For the purpose of this study, the subjects were divided into the pretreatment group (n = 28), the posttreatment group (n = 28), and the control group (n = 28). The diagnosis of DOR is done via expert consensus on hormone replacement therapy for premature ovarian insufficiency [For the pseudometabolomics analysis, the MetSizeR approach for sample size estimation was used to estimate a total sample size of 84 subjects using the following assumptions: 954 follicular fluid metabolites, a target false detection rate of 5%, and an expected proportion of significant metabolites of 20% . SubjectInclusion criteria of the DOR patients are as follows: (1) kidney-yin deficiency of traditional Chinese medicine; (2) 20–40-year-old; (3) 10 IU/ml ＜ FSH ＜ 25 IU/ml; (4) patients of the control group who had a history of induced abortions of a normal pregnancy.Inclusion criteria of the control group are as follows: (1) infertility due to male factor; (2) 20–40-year-old.Exclusion criteria are as follows: (1) congenital genital dysplasia; (2) cerebrovascular and cardiovascular; (3) hyperprolactinemia and polycystic ovary syndrome; (4) fail to receive treatment as required due to personal and other reasons; (5) poor treatment effect.μL were mixed with 300 μL of methanol containing 500 ng/mL of internal standards. The mixture was vortexed for 2 min and then centrifuged at 13000 ×g for 20 min, at 4°C. The supernatant was then transferred to an autosampler plate for analysis.Follicular fluid samples (approximately 1.0 mL) were collected according to the previous protocol . Follicu∗ 2.1 mm, 1.7 μm) column  at 40°C. Analysis was completed with a gradient elution of 0.1% formic acid in acetonitrile (A) and 10 mM ammonium acetate in water (B) within 10.0 min. The gradient program was 5% B at 0.0–0.3 min, 95% BA at 5.0–8.0 min, and 5% B at 8.1–10.0 min at a flow rate of 0.4 mL/min with a sample injection volume of 10.0 μL. All the samples were kept at 4°C during the analysis.The separation was performed using a SCIEX ExionLC AD ultraperformance liquid chromatography system , while the chromatographic separation was performed on a Waters HILIC . The multireaction monitor (MRM) and enhanced production (EPI) data were acquired in positive/negative electrospray ionization mode with dynamic background subtraction (DBS). The MRM transitions were extracted from the follicular fluid MS/MS spectra, which were acquired from TripleTOF5600 . Source parameters were defined as follows: temperature, 550°C; ion spray voltage, 5500 V (positive)/-4500 V (negative); nebulizer gas (gas 1), 60 psi; heater gas (gas 2), 60 psi; curtain gas, 30 psi; and declustering potential, 60 V. For IDA, any MRM survey scan peak exceeding 150 cps was selected for dependent scan. 5 candidate ions were allowed per cycle. The collision energy was set to 35 V (positive)/-35 V (negative) with a collision energy spread of 15 V. All operations and data acquisition were controlled by the Analyst 1.6 software .p value cutoff 0.05 in one-way ANOVA and VIP score > 1 were considered as potential biomarkers. The software used for other statistical analysis was MetaboAnalyst software 4.0 .The LC-MS data files were imported into MQ software . The chromatographic peaks were extracted, respectively, while Pareto scaling and multivariate analyses of log transformation were applied to the data processing before LPS-DA was performed to give the contribution of the metabolites by VIP score. All the compounds with significance threshold satisfying corrected http://www.hmdb.ca).OS software  was used to help confirm the potential differential metabolites by analyzing with the fragmentation patterns, chromatographic retention characteristics, and comparing the mass-spectra with the authentic standards. The fragmentation of the potential metabolites was matched with the metabolites from online databases HMDB  among the posttreatment group, the pretreatment group, and the control group. The results are shown in There was no significant difference in age, infertility years, body mass index, basic LH, basic E2, among basic p < 0.001) (seen in p < 0.05). It is suggested that LWDH pill can improve the syndrome of kidney-yin deficiency of DOR patients. The results are shown in The total syndrome integral of DOR patients decreased significantly after treatment using LWDH pill ( seen in . The synp > 0.05). The number of oocytes (<14 mm follicles or ≥ 14 mm follicles) was statistically different (p < 0.05) during the day of hCG between pretreatment and posttreatment, which indicated that LWDH pill could improve the ovarian reserve of DOR patients and effectively stimulate the growth of follicles. The results showed that there were significant differences between pretreatment and posttreatment in the number of transplantable embryos and IVF fertilization rate (p < 0.05). However, there was no significant difference in the level of E2, the level of P, the number of high-quality embryos, the oocyte acquisition rate, and the clinical pregnancy rate (p > 0.05). The detail information is shown in There was no significant difference in hCG daily endocrine between the pretreatment and posttreatment propyl disulfide, eicosatrienoic acid, 20α-dihydroprogesterone, 17-beta-estradiol-3,17-beta-sulfate, tryptophan, lactate, oxaloacetate, and O-decanoyl-L-carnitine upregulated. After the treatment of LWDH, the level of all 21 biomarkers in follicular fluid significantly backregulated comparing with the pretreatment group. The contribution of each potential biomarker to the discrimination between the two groups was ranked as VIP scores shown in A large-scale multireaction monitor (MRM) was applied in the LC-MS detection. Typical chromatograms of follicular fluid are shown in The metabolic pathways related to the identified potential biomarkers were estimated by pathway topology analysis using MetaboAnalyst based on the KEGG reference pathways, in order to find possible metabolic pathways that have great impact on DOR. A figure of metabolic pathway impact which demonstrated the pathway impact values is shown in The clinical manifestations of DOR were including low menstrual volume, irregular menstruation, and infertility. Patients with DOR often have symptoms of deficiency of yin and essence, such as palpitation, sweating, irritability, insomnia, and forgetfulness. As the most basic essence of human life, the kidney stores essence. When it is sufficient, women can be gestated by menstruation. With the increase in age, kidney essence gradually decreased, and reproductive function declined. LWDH pill could fill lean marrow and nourish yin and kidney. The integral of the posttreatment group was significantly lower than that of the pretreatment group, seen in Choline is essential for acetylcholine biosynthesis. Acetylcholine is a key neurotransmitter for neuron differentiation and maturation. A large intake of choline during pregnancy is beneficial to the development of embryo and the cognitive function of offspring , 22. DurPyruvic acid is a kind of weak organic acid. It is the intermediate substance of Chinese medicine for the synthesis and decomposition of many substances, including oxidation metabolism, gluconeogenesis pathway, tricarboxylic acid cycle, and lipid synthesis. Lactic acid is the metabolite of glycolysis, which is closely related to energy metabolism, lipid metabolism, protein metabolism, and glucose metabolism. It was found that glucose and pyruvate affect the cytoplasmic maturation of porcine oocytes through glycolysis. Pyruvate can improve ROS, GSH, ATP, and early embryonic development of MII oocytes and promote the cytoplasmic maturation of pigs by providing energy and reducing oxidative stress , 38. In Testosterone is a kind of anabolic steroid hormone. It plays an important role in ovarian response through androgen receptor (AR) pathway and non-AR pathway. It can induce the activation of primordial follicles by inhibiting PTEN expression via the AR pathway . The incβ-estradiol-3 and 17β-sulfate are the metabolites of estrogen. Estradiol is closely related to the fertilization ability of oocytes, and its metabolites could stimulate the production of progesterone. However, if the level of estradiol is too high, it can destroy the blood flow of uterus and ovary and may lead to anoxia of follicles to damage the development potential of embryos. Jiang et al. found that women with high estradiol levels had poor pregnancy outcomes [p < 0.05).Luteal hormone is a hormone secreted by the ovary, which can inhibit the proliferation of granulosa cells induced by epidermal growth factor. It affects cell oxidative stress . 8-Hydrooutcomes . In our α (TNF) is a kind of cytokine, which is produced not only by various cells in the immune system but also by various cells in the reproductive system. Indole is produced by the metabolism of tryptophan [Tryptophan may affect oocyte development and follicle quality through immunity. It can be transformed into melatonin. Melatonin can delay the aging of mouse oocytes after ovulation through SIRT1 MnSOD-dependent pathway , which cyptophan . Indole yptophan . The absyptophan .13,14-Dihydroretinol is related to retinoic acid metabolism. Retinoic acid is the intermediate metabolite of vitamin A. Some studies have found that vitamin A can protect ROS-induced cell oxidative stress . 13,14-Dp-Cresol sulfate is a biomarker of renal decline [Acrylamide has toxic effects on the thyroid, red blood cells, skeletal muscle, smooth muscle, and nervous system. At the same time, it will affect the proliferation of mouse granulosa cells and the production of progesterone , resulti decline . Some st decline . MitochoMetabolic changes are relevant to many diseases. In previous study, lipidomics has contributed an improved understanding of a potential underlying mechanism of ovarian aging and poor oocyte quality. Lipids seem to play a crucial role in ovary aging, and further analysis and validation of potential lipid biomarkers may facilitate improved IVF in the future . In our A pseudotargeted metabolomics profiling on the follicular fluid was successfully established and integrally investigated. The follicular fluid metabolome was obviously altered in DOR patients after LWDH pill treatment. After the pseudotarget metabolomics screening, a total of 21 metabolic biomarker candidates, which may be related to DOR were characterized, and their correlative metabolomic pathways were revealed. In addition, it was speculated that the potential active targets of LWDH pill on DOR may be more comprehensive than chemical treatments. The study demonstrated that the metabolomics strategy might serve as a powerful approach for investigating the mechanisms of DOR and LWDH pills using the processing method, while providing solutions to explore the molecular basis of diseases and identify potential biomarkers.


Enterobacteriaceae from different sources of samples from Khartoum state, Sudan, and to determine the prevalence rate of extended-spectrum beta-lactamase (ESBL), multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacteria. A total of 144 Gram-negative bacteria were collected from different sources . Samples were subcultured and identified according to their cultural characteristics and biochemical tests. Antimicrobial susceptibility test was performed for twenty-four antibiotics from eleven categories against all isolated Enterobacteriaceae according to the recommendation of Clinical and Laboratory Standards Institute (CLSI). The result showed that out of 144 isolates, Escherichia coli and Klebsiella pneumoniae were predominant isolates with the percentage of 47.9 and 25%, respectively. The prevalence of ESBL was higher in K. pneumonia (38.9%) than E. coli (34.8%). All isolated E. coli were sensitive to nitrofurantoin and tigecycline. There was a high prevalence of MDR Enterobacteriaceae, and only one isolate was XDR, while PDR was zero for all isolated bacteria. Active antimicrobial-resistant (AMR) observation through constant data sharing and management of all stakeholders is crucial to recognize and control the AMR global burden. Also, effective antibiotic stewardship procedures would be applied to limit the unreasonable expenditure of antibiotics in Sudan.One of the global requirements for controlling the occurrence of resistance to antimicrobial drugs is to understanding the resistivity profile of various clinical isolates. Therefore, this study aimed to deliver the indication of different resistant profiles of clinically isolated  Escherichia coli and Klebsiella spp. had touched frightening levels of resistance in various parts of the world including resistant to third-generation cephalosporins and carbapenems of up to 54% [The prevalence of multidrug-resistant bacterial infection has been increasing globally, which is exacerbated due to the scarcity of innovative classes of antibiotics tested clinically during the past 40 years . BacteriFurthermore, information of the resident bacterial etiology and resistance profile is essential to observe any modification that may be happened within time. Therefore, optimum empirical therapy can be achieved by continuous updating of antibiogram , 6. The Enterobacteriaceae in clinical samples and screen the antibiotics profile to the most regularly used antimicrobials and to determine the occurrence rates of extended-spectrum beta-lactamase (ESBL), multidrug resistance (MDR), extensive drug resistance (XDR), and pandrug resistance (PDR).From all of the above, worldwide epidemiological observation is critical to comprehensive AMR reaction. Understanding of indigenous and regional AMR is significant for medical choices creation. Still, exploration capability for AMR needs throughout Sudan, and existing AMR records are little, as no full data have been described from the present study area. Therefore, this study aimed to determine the prevalence of This was a cross-sectional study conducted over 6 months from February to July 2018. A total of one hundred and forty-four isolated Gram-negative bacteria from different clinical sample sources  were collected from two main tertiary care referral hospitals in the Khartoum state . All Gram-negative isolated bacteria in the microbiology laboratory were included in this study. There are no exclusion criteria.Isolated Gram-negative bacteria were subcultured on nutrient agar and incubated aerobically at 37°C for 24 hours and then identified according to the culture characteristics on Xylose Lysine Deoxycholate agar (XLD), MacConkey agar, and biochemical tests .β-lactam/β-lactamase inhibitor complexes, folate metabolic pathway inhibitors, glycylcyclines, cephamycins, and nitrofuran as recommended for Gram-negative bacteria [μg), cefuroxime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), moxifloxacin (5 μg), levofloxacin (5 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), tobramycin (10 μg), gentamicin (10 μg), amikacin (30 μg), ampicillin (10 μg), cefoxitin (30 μg), aztreonam (30 μg), amoxicillin-clavulanic acid (30 μg), ampicillin-sulbactam (10/10 μg), trimethoprim/sulfamethoxazole (5/250 μg), piperacillin-tazobactam (100/10 μg), nitrofurantoin (300 μg), and tigecycline (15 μg) were used according to the standard procedures (CLSI). The results were interpreted according to the recommendation of the Clinical Laboratory Standards Institute [Various categories of antibiotics were used in this study included cephalosporins, fluoroquinolones, carbapenems, aminoglycosides, penicillins, monobactams, bacteria . The antnstitute .E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and K. pneumoniae ATCC BAA700603 were used.For the reliability of study findings, quality control implements measures of performance checks during the entire procedure of the laboratory work. β-lactamase (ESBL) was recognized as the resistance rate against cefotaxime and ceftazidime. MDR, XDR, and PDR were calculated in this study for each isolated bacteria as described by Magiorakos et al. [The prevalence of extended-spectrum s et al. .P value of ≤0.05 for the association bacteria and source of samples was considered significant.Data were analyzed using Statistical Package for Social Sciences (SPSS), version 25 . Descriptive data were expressed as a percentage. A E. coli and K. pneumonia were predominant with percentages 69 (47.9%) and 36 (25%), respectively. However, Salmonella enterica was 5 (3.47%), Shigella spp. was 1 (0.69%), Citrobacter freundii was 1 (0.69%), Klebsiella oxytoca was 3 (2.08%), Enterobacter cloacae was 1 (0.69%), Proteus mirabilis was 3 (2.08%), and Enterobacter aerogenes was 2 (1.38%).Out of 144 Gram-negative bacterial isolates, Enterobacteriaceae were from urine with a frequency of 51 (35.4%) and 22 (15.37%) for E. coli and K. pneumonia, respectively. There was a significant association between isolated bacteria and sources of the sample, with P value = 0.001 , while antibiotics from the cephalosporins class exhibited a high resistance rate among Enterobacteriaceae bacteria, with the resistance rate for E. coli towards ceftazidime (39.1%), and cefotaxime and cefuroxime (37.7%). However, among the drugs belonging to the fluoroquinolones classes, the highest percentage of the resistant rate was obtained by E. coli for ciprofloxacin (18.8%) and levofloxacin (17.4%). The lowest resistant rate was obtained for carbapenems drugs with the E. coli resistant percentage of 1.4% for imipenem and meropenem. Moreover, all isolated E. coli were sensitive towards nitrofurantoin and tigecycline; the resistant rate was zero (Most of the isolated  = 0.001 . Regardiwas zero .β-lactamase producer ESBL among E. coli and K. pneumonia (E. cloacae 1(100%) which was sensitive only to amikacin and piperacillin-tazobactam, while PDR was zero for all isolated bacteria.There was a high prevalence of MDR bacteria and extended-spectrum neumonia . XDR wasEnterobacteriaceae as a causative agent for infections, as well as the resistant rate towards various antibiotics belonging to different categories, and the frequency of ESBL, MDR, XDR and PDR were determined.This study reported the prevalence of Gram-negative E. coli and K. pneumonia were the major isolated bacteria from urine samples which mean the leading causative agent for urinary tract infection (UTI). These results entirely agreed with other studies which found that these bacteria were also predominantly causing UTI [E. coli and K. pneumonia which exhibited a high resistant rate  to cefotaxime and ceftazidime were classified as extended-spectrum β-lactamase (ESBL) phenotypes. This ESBL prevalence rate was near to the rate reported in the previous studies from Sudan for E. coli and K. pneumonia (28.9% and 34.8%) [In the current study, sing UTI –15. The d 34.8%) , and in d 34.8%) –21.Enterobacteriaceae and represents an alarming threat of appearance of multidrug-resistant pathogens.In general, the increase in the resistance of isolated organisms to penicillin, fluoroquinolones, macrolide, and cephalosporins in this study might be due to the increase in the usage of these antibiotic's classes in the hospital. Also, our study presented the high MDR bacteria, which showed resistance to more than three groups of tested antibiotics. This MDR rate entirely agrees with other studies in Sudan which reported a high prevalence of MDR bacteria , 23. ThiE. coli and K. pneumonia isolates from different samples sources. Some other correlated reports pointed out carelessness from the patient's part, tentative treatment plans, self-prescription, antibiotics misuse, misprescription, and limited information around multidrug-resistant isolates and antimicrobial resistance among clinicians. Nevertheless, the study was conducted in only one state; however, this study will update the records of resistant rates of clinical isolates. Also, it will capture the responsiveness of all hospitals' controlling team in making proper assessments and research to manage the progress of the resistant strain and also help to decrease the alarmingly increasing risk of drug resistance.The current study delivers the confirmation of high occurrence of antibiotic-resistant bacteria in urine, tracheal aspirate, and vaginal swab samples. Also, there is a presence of high drug resistance to various antibiotics in Enterobacteriaceae from different clinical samples from Khartoum state, Sudan. Therefore, the result of this study may be proof of the urgent need for controlling and managing the development of MDR strain. Moreover, antibiotic stewardship procedures should be applied to limit the illogical use of antibiotics in Sudan.In conclusion, this study highlighted the antibiotics profile and prevalence of ESBL, MDR, XDR, and PDR among Our perspective cross-sectional study has some limitations that should be recognized, which it was conducted in one state (Sudan consists of 18 states), which could not reflect the epidemiology of different states or different geographical areas.