Detection of viruses in clinical samples by use of metagenomic sequencing and targeted sequence capture
^{Wylie KM, Wylie TN, Buller R, Herter B, Cannella MT, Storch GA. J Clin Microbiol 2018; 56(12): pii: e01123-1}

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyse clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analysed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.

Sonication versus tissue sampling for diagnosis of prosthetic joint and other orthopedic device-related infections
^{Dudareva M, Barrett L, Figtree M, Scarborough M, Watanabe M, et al. J Clin Microbiol 2018; 56(12): pii: e00688-18}

Current guidelines recommend collection of multiple tissue samples for diagnosis of prosthetic joint infections (PJI). Sonication of explanted devices has been proposed as a potentially simpler alternative; however, reported microbiological yield varies. We evaluated sonication for diagnosis of PJI and other orthopedic device-related infections (DRI) at the Oxford Bone Infection Unit between October 2012 and August 2016. We compared the performance of paired tissue and sonication cultures against a ‘gold standard’ of published clinical and composite clinical and microbiological definitions of infection. We analysed explanted devices and a median of five tissue specimens from 505 procedures. Among clinically infected cases the sensitivity of tissue and sonication culture was 69% (95% confidence interval, 63 to 75) and 57% (50 to 63), respectively (P<0.0001). Tissue culture was more sensitive than sonication for both PJI and other DRI, irrespective of the infection definition used. Tissue culture yield was higher for all subgroups except less virulent infections, among which tissue and sonication culture yield were similar. The combined sensitivity of tissue and sonication culture was 76% (70 to 81) and increased with the number of tissue specimens obtained. Tissue culture specificity was 97% (94 to 99), compared with 94% (90 to 97) for sonication (P=0.052) and 93% (89 to 96) for the two methods combined. Tissue culture is more sensitive and may be more specific than sonication for diagnosis of orthopedic DRI in our setting. Variable methodology and case mix may explain reported differences between centres in the relative yield of tissue and sonication culture. Culture yield was highest for both methods combined.

MODS-Wayne, a colorimetric adaptation of the microscopic-observation drug susceptibility (MODS) assay for detection of Mycobacterium tuberculosis pyrazinamide resistance from sputum samples
^{Alcántara R, Fuentes P, Antiparra R, Santos M, Gilman RH, et al. J Clin Microbiol 2019; 57(2): pii: e01162-18}

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a colour change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/mL, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.

Barriers and facilitators and the need for a clinical guideline for microbiological diagnostic testing in the hospital: a qualitative and quantitative study
^{Bogers SJ, van Daalen FV, Kuil SD, de Jong MD, Geerlings SE. Eur J Clin Microbiol Infect Dis 2019; doi: 10.1007/s10096-019-03516-z [Epub ahead of print]}

The appropriate use of microbiological investigations is an important cornerstone of antibiotic stewardship programmes, but receives relatively limited attention. This study aimed to identify influencing factors in performing microbiological diagnostic tests and to assess the need for a clinical guideline. We performed a qualitative (focus group) and quantitative (online questionnaire survey) study among medical specialists and residents to identify physicians’ considerations in performing microbiological diagnostic tests and to assess the need for a diagnostic guideline. The questionnaire consisted of 14 statements, divided into three categories: knowledge, influencing factors and presence of guidelines. The questionnaire was sent to physicians of the departments of internal medicine, intensive care, pediatrics and pulmonology in five hospitals in the Netherlands. Sub-analyses for medical specialists versus residents and for pediatric versus non-pediatric departments were performed. We included 187 completed questionnaires in our analyses. The physicians reported having adequate knowledge on methods, time-to-result and accuracy, but inadequate knowledge on costs of the tests. Patients’ clinical condition, comorbidity, local guidelines and accuracy of tests were appraised as the four most important influencing factors to perform tests. Over 70% (132/187) of physicians reported being interested in a guideline for microbiological diagnostic testing. Fifteen physicians (8.0%) provided additional comments. This study identifies the influencing factors to microbiological testing and shows the demand for a clinical guideline among physicians. IMPORTANCE: Microbiological diagnostic tests are an important cornerstone within antibiotic stewardship programmes. These programmes aim to ameliorate the appropriate use of antibiotics and thus improve clinical outcomes of infectious diseases, whilst reducing the emergence of antimicrobial resistance. However, inappropriate microbiological testing is a widely recognized problem, and influencing factors to testing have not been studied in the past. Our research shows the demand for a clinical guideline among physicians, and it identifies their influencing factors to testing. These results can be used to create a clinical guideline for microbiological diagnostic testing, thus supporting antibiotic stewardship programmes and reducing antimicrobial resistance.

Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory
^{Boers SA, Jansen R, Hays JP. Eur J Clin Microbiol Infect Dis 2019; doi: 10.1007/s10096-019-03520-3 [Epub ahead of print]}

Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of (‘difficult-to-culture’) microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics, however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e. targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform (‘MYcrobiota’) is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.

A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing
^{Walker A, Ennker KS, Kaiser R, Lübke N, Timm J. J Clin Virol 2019; 113: 8–13}

BACKGROUND: Chronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved; however, the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success.

OBJECTIVE: The aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup.

STUDY DESIGN: Pan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1–7 and the median viral load was 1.07×106 IU/mL (range 248–21×106 IU/mL).

RESULTS: Amplification of the expected 747 bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/mL. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples.

CONCLUSIONS: We successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.

Impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections
^{Lee BR, Hassan F, Jackson MA, Selvarangan R. J Clin Virol 2019; 110: 11–16}

BACKGROUND: Empiric antibiotic treatment is common among children with acute respiratory tract infections (ARTI), despite infections being predominately viral. The use of molecular respiratory panel assays has become increasingly common for medical care of patients with ARTIs.

STUDY DESIGN: This was a 6-year retrospective, single-centred study of pediatric inpatients who tested positive for an ARTI respiratory pathogen. We examined the relationship between clinical outcomes and whether the patient was tested using the Luminex Respiratory Viral Panel ([RVP]; in-use: Dec 2009 – Jul 2012) or Biofire Respiratory Pathogen Panel ([RP]; in-use Aug 2012 – Jun 2016). The prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest X-rays and length of stay between the two assays was compared.

RESULTS: A total of 5142 patients (1264 RVP; 3878 RP) were included. The median laboratory turn-around-time for RP was significantly shorter than RVP (1.4 vs 27.1 h, respectively; P<0.001). Patients tested with RP were less likely to receive empiric antibiotics (OR: 0.45; P<0.001; 95% CI: 0.39, 0.52) and had a shorter duration of empiric broad-spectrum antibiotics (6.4 h vs 32.9 h; P<0.001) compared to RVP patients. RP influenza patients had increased oseltamivir use post- test compared to RVP influenza patients (OR: 13.56; P<0.001; 95% CI: 7.29, 25.20).

CONCLUSIONS: Rapid molecular testing positively impacts patient management of ARTIs. Adopting assays with a shorter turn-around-time improves decision making by decreasing empirical antibiotic use and duration, decreasing chest X-rays, increasing timely oseltamivir administration, and reducing length of stay.

Practical guidance for clinical microbiology laboratories: viruses causing acute respiratory tract infections
^{Charlton CL, Babady E, Ginocchio CC, Hatchette TF, Jerris RC, et al. Clin Microbiol Rev 2018; 32(1): pii: e00042-18}

Respiratory viral infections are associated with a wide range of acute syndromes and infectious disease processes in children and adults worldwide. Many viruses are implicated in these infections, and these viruses are spread largely via respiratory means between humans but also occasionally from animals to humans. This article is an American Society for Microbiology (ASM)-sponsored Practical Guidance for Clinical Microbiology (PGCM) document identifying best practices for diagnosis and characterization of viruses that cause acute respiratory infections and replaces the most recent prior version of the ASM-sponsored Cumitech 21 document, Laboratory Diagnosis of Viral Respiratory Disease, published in 1986. The scope of the original document was quite broad, with an emphasis on clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen detection and viral culture. The new PGCM document is designed to be used by laboratorians in a wide variety of diagnostic and public health microbiology/virology laboratory settings worldwide. The article provides guidance to a rapidly changing field of diagnostics and outlines the epidemiology and clinical impact of acute respiratory viral infections, including preferred methods of specimen collection and current methods for diagnosis and characterization of viral pathogens causing acute respiratory tract infections. Compared to the case in 1986, molecular techniques are now the preferred diagnostic approaches for the detection of acute respiratory viruses, and they allow for automation, high-throughput workflows, and near-patient testing. These changes require quality assurance programs to prevent laboratory contamination as well as strong preanalytical screening approaches to utilize laboratory resources appropriately. Appropriate guidance from laboratorians to stakeholders will allow for appropriate specimen collection, as well as correct test ordering that will quickly identify highly transmissible emerging pathogens.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the rapid detection of antimicrobial resistance mechanisms and beyond
^{Oviaño M, Bou G. Clin Microbiol Rev 2018; 32(1): pii: e00037-18}

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied in recent years for first-line identification of pathogens in clinical microbiology because it is simple to use, rapid, and accurate and has economic benefits in hospital management. The range of clinical applications of MALDI-TOF MS for bacterial isolates is increasing constantly, from species identification to the two most promising applications in the near future: detection of antimicrobial resistance and strain typing for epidemiological studies. The aim of this review is to outline the contribution of previous MALDI-TOF MS studies in relation to detection of antimicrobial resistance and to discuss potential future challenges in this field. Three main approaches are ready (or almost ready) for clinical use, including the detection of antibiotic modifications due to the enzymatic activity of bacteria, the detection of antimicrobial resistance by analysis of the peak patterns of bacteria or mass peak profiles, and the detection of resistance by semiquantification of bacterial growth in the presence of a given antibiotic. This review provides an expert guide for MALDI-TOF MS users to new approaches in the field of antimicrobial resistance detection, especially possible applications as a routine diagnostic tool in microbiology laboratories.

Skin autofluorescence predicts incident type 2 diabetes, cardiovascular disease and mortality in the general population
^{van Waateringe RP, Fokkens BT, Slagter SN, van der Klauw MM, van Vliet-Ostaptchouk JV, et al. Diabetologia 2019; 62(2): 269–280}

AIMS/HYPOTHESIS: Earlier studies have shown that skin autofluorescence measured with an AGE reader estimates the accumulation of AGEs in the skin, which increases with ageing and is associated with the metabolic syndrome and type 2 diabetes. In the present study, we examined whether the measurement of skin autofluorescence can predict 4-year risk of incident type 2 diabetes, cardiovascular disease (CVD) and mortality in the general population.

METHODS: For this prospective analysis, we included 72  880 participants of the Dutch Lifelines Cohort Study, who underwent baseline investigations between 2007 and 2013, had validated baseline skin autofluorescence values available and were not known to have diabetes or CVD. Individuals were diagnosed with incident type 2 diabetes by self-report or by a fasting blood glucose ≥7.0 mmol/L or HbA1c ≥48 mmol/mol (≥6.5%) at follow-up. Participants were diagnosed as having incident CVD (myocardial infarction, coronary interventions, cerebrovascular accident, transient ischaemic attack, intermittent claudication or vascular surgery) by self-report. Mortality was ascertained using the Municipal Personal Records Database.

RESULTS: After a median follow-up of 4 years (range 0.5–10 years), 1056 participants (1.4%) had developed type 2 diabetes, 1258 individuals (1.7%) were diagnosed with CVD, while 928 (1.3%) had died. Baseline skin autofluorescence was elevated in participants with incident type 2 diabetes and/or CVD and in those who had died (all P< 0.001), compared with individuals who survived and remained free of the two diseases. Skin autofluorescence predicted the development of type 2 diabetes, CVD and mortality, independent of several traditional risk factors, such as the metabolic syndrome, glucose and HbA1c.

CONCLUSIONS/INTERPRETATION: The non-invasive skin autofluorescence measurement is of clinical value for screening for future risk of type 2 diabetes, CVD and mortality, independent of glycaemic measures and the metabolic syndrome.

A renal genetic risk score (GRS)  is associated with kidney dysfunction in people with type 2 diabetes
^{Zusi C, Trombetta M, Bonetti S, Dauriz M, Boselli ML, et al. Diabetes Res Clin Pract 2018; 144: 137–143}

This study aims to investigate whether renal and cardiovascular phenotypes in Italian patients with type 2 diabetes (T2D) could be influenced by a number of disease risk SNPs recently found in genome-wide association studies (GWAS). In 1591 Italian subjects with T2D: (1) 47SNPs associated to kidney function and/or chronic kidney disease (CKD) and 49SNPs associated to cardiovascular disease (CVD) risk were genotyped; (2) urinary albumin/creatinine (A/C) ratio, glomerular filtration rate (eGFR) and lipid profile were assessed; (3) a standard electrocardiogram was performed; (4) two genotype risk scores (GRS) were computed (a renal GRS calculated selecting 39 SNPs associated with intermediate traits of kidney damage and a cardiovascular GRS determined selecting 42 SNPs associated to CVD risk phenotypes). After correction for multiple comparisons, the renal GRS was not associated to A/C ratio (P=0.33), but it was significantly related to decreased eGFR (P=0.005). No association between the cardiovascular GRS and electrocardiogram was detected. Thus, in Italian patients with T2D a renal GRS might predict the decline in glomerular function, suggesting that the clock of diabetes associated CKD starts ticking long before hyperglycemia. Our data support the feasibility of gene-based prediction of complications in people with T2D.

Protein markers and risk of type 2 diabetes and prediabetes: a targeted proteomics approach in the KORA F4/FF4 study
^{Huth C, von Toerne C, Schederecker F, de Las Heras Gala T, Herder C, et al. Eur J Epidemiol 2018: doi: 10.1007/s10654-018-0475-8 [Epub ahead of print]}

The objective of the present study was to identify proteins that contribute to pathophysiology and allow prediction of incident type 2 diabetes or incident prediabetes. We quantified 14 candidate proteins using targeted mass spectrometry in plasma samples of the prospective, population-based German KORA F4/FF4 study (6.5-year follow-up). 892 participants aged 42–81 years were selected using a case-cohort design, including 123 persons with incident type 2 diabetes and 255 persons with incident WHO-defined prediabetes. Prospective associations between protein levels and diabetes, prediabetes as well as continuous fasting and 2 h glucose, fasting insulin and insulin resistance were investigated using regression models adjusted for established risk factors. The best predictive panel of proteins on top of a non-invasive risk factor model or on top of HbA1c, age and sex was selected. Mannan-binding lectin serine peptidase (MASP) levels were positively associated with both incident type 2 diabetes and prediabetes. Adiponectin was inversely associated with incident type 2 diabetes. MASP, adiponectin, apolipoprotein A-IV, apolipoprotein C-II, C-reactive protein, and glycosylphosphatidylinositol specific phospholipase D1 were associated with individual continuous outcomes. The combination of MASP, apolipoprotein E (apoE) and adiponectin improved diabetes prediction on top of both reference models, while prediabetes prediction was improved by MASP plus CRP on top of the HbA1c model. In conclusion, our mass spectrometric approach revealed a novel association of MASP with incident type 2 diabetes and incident prediabetes. In combination, MASP, adiponectin and apoE improved type 2 diabetes prediction beyond non-invasive risk factors or HbA1c, age and sex.

Association between circulating tumor necrosis factor-related biomarkers and estimated glomerular filtration rate in type 2 diabetes.
^{Kamei N, Yamashita M, Nishizaki Y, Yanagisawa N, Nojiri S, et al. Sci Rep 2018; 8(1): 15302}

Chronic inflammation plays a crucial role in the development/progression of diabetic kidney disease. The involvement of tumor necrosis factor (TNF)-related biomarkers [TNFα, progranulin (PGRN), TNF receptors (TNFR1 and TNFR2)] and uric acid (UA) in renal function decline was investigated in patients with type 2 diabetes (T2D). Serum TNF-related biomarkers and UA levels were measured in 594 Japanese patients with T2D and an eGFR ≥30 mL/min/1.73 m². Four TNF-related biomarkers and UA were negatively associated with estimated glomerular filtration rate (eGFR). In a logistic multivariate model, each TNF-related biomarker and UA was associated with lower eGFR (eGFR <60 mL/min/1.73 m²) after adjustment for relevant covariates (basic model). Furthermore, UA and TNF-related biomarkers other than PGRN added a significant benefit for the risk factors of lower eGFR when measured together with a basic model (UA, ΔAUC, 0.049, P<0.001; TNFα, ΔAUC, 0.022, P=0.007; TNFR1, ΔAUC, 0.064, P<0.001; TNFR2, ΔAUC, 0.052, P<0.001) in receiver operating characteristic curve analysis. TNFR ligands were associated with lower eGFR, but the associations were not as strong as those with TNFRs or UA in patients with T2D and an eGFR ≥30 mL/min/1.73 m².

Plasma endostatin predicts kidney outcomes in patients with type 2 diabetes
^{Chauhan K, Verghese DA, Rao V, Chan L, Parikh CR, et al. Kidney Int 2019; 95(2): 439–446}

Novel biomarkers are needed to predict kidney function decline in patients with type 2 diabetes, especially those with preserved glomerular filtration rate (GFR). There are limited data on the association of markers of endothelial dysfunction with longitudinal GFR decline. We used banked specimens from a nested case-control study in the Action to Control Cardiovascular Disease (ACCORD) trial (n=187 cases; 187 controls) and from a diverse contemporary cohort of type 2 diabetic patients from the Mount Sinai BioMe Biobank (n=871) to assess the association of plasma endostatin and kidney outcomes. We measured plasma endostatin at enrolment and examined its association with a composite kidney outcome of sustained 40% decline in estimated GFR or end-stage renal disease. Baseline plasma endostatin levels were higher in participants with the composite outcome. Each log2 increment in plasma endostatin was associated with approximately 2.5-fold higher risk of the kidney outcome (adjusted odds ratio [OR] 2.5; 95% confidence interval [CI] 1.5–4.3 in ACCORD and adjusted hazard ratio [HR] 2.6; 95% CI 1.8-3.8 in BioMe). Participants in the highest versus lowest quartile of plasma endostatin had approximately fourfold higher risk for the kidney outcome (adjusted OR 3.6; 95% CI 1.8-7.3 in ACCORD and adjusted HR 4.4; 95% CI 2.3-8.5 in BioMe). The AUC for the kidney outcome improved from 0.74 to 0.77 in BioMe with the addition of endostatin to a base clinical model. Plasma endostatin was strongly associated with kidney outcomes in type 2 diabetics with preserved eGFR and improved risk discrimination over traditional predictors

Relation of serum and urine renal biomarkers to cardiovascular risk in patients with type 2 diabetes mellitus and recent acute coronary syndromes (from the EXAMINE Trial)
^{Vaduganathan M, White WB, Charytan DM, Morrow DA, Liu Y, et al. Am J Cardiol 2019; 123(3): 382–391}

A deeper understanding of the interplay between the renal axis and cardiovascular (CV) disease is needed in type 2 diabetes mellitus (T2DM). We aimed to explore the prognostic value of a comprehensive panel of renal biomarkers in patients with T2DM at high CV risk. We evaluated the prognostic performance of both serum (Cystatin C) and urine renal biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 protein, and indices of urinary protein excretion) in 5380 patients with T2DM and recent acute coronary syndromes in the EXAMINE trial. Patients requiring dialysis within 14 days were excluded. Single- and multimarker covariate-adjusted Cox proportional hazards models were developed to predict times to events. Primary endpoint was composite nonfatal myocardial infarction, nonfatal stroke, or CV death. Median age was 61 years, 68% were men, and mean baseline estimated glomerular filtration rate (eGFR) was 74 mL/min/1.73 m². During median follow-up of 18 months, 621 (11.5%) experienced the primary endpoint and 326 (6.1%) patients had died. All renal biomarkers were robustly associated with adverse CV events in step-wise fashion, independent of baseline eGFR. However, in the multimarker prediction model, only Cystatin C (per 1 SD) was associated with the primary endpoint (hazard ratio [HR] 1.28 [1.14 to 1.45]; P≤0.001), death (HR 1.51 [1.30 to 1.74]; P≤0.001), and heart failure hospitalization (HR 1.20 [0.96 to 1.49]; P=0.11). Association between Cystatin C and the primary endpoint was similar in baseline eGFR above and below 60 mL/min/1.73 m² (P_interaction >0.05). In conclusion, serum and urine renal biomarkers, when tested alone, independently predict long-term adverse CV events in high-risk patients with T2DM. In an integrative panel of renal biomarkers, only serum Cystatin C remained independently associated with subsequent CV risk. Renal biomarkers informing various aspects of kidney function may further our understanding of the complex interplay between diabetic kidney disease and CV disease.

A plasma circulating miRNAs profile predicts type 2 diabetes mellitus and prediabetes: from the CORDIOPREV study
^{Jiménez-Lucena R, Camargo A, Alcalá-Diaz JF, Romero-Baldonado C, Luque RM, et al. Exp Mol Med 2018; 50(12): 168}

We aimed to explore whether changes in circulating levels of miRNAs according to type 2 diabetes mellitus (T2DM) or prediabetes status could be used as biomarkers to evaluate the risk of developing the disease. The study included 462 patients without T2DM at baseline from the CORDIOPREV trial. After a median follow-up of 60 months, 107 of the subjects developed T2DM, 30 developed prediabetes, 223 maintained prediabetes and 78 remained disease-free. Plasma levels of four miRNAs related to insulin signalling and beta-cell function were measured by RT-PCR. We analysed the relationship between miRNAs levels and insulin signalling and release indexes at baseline and after the follow-up period. The risk of developing disease based on tertiles (T1-T2-T3) of baseline miRNAs levels was evaluated by Cox analysis. Thus, we observed higher miR-150 and miR-30a-5p and lower miR-15a and miR-375 baseline levels in subjects with T2DM than in disease-free subjects. Patients with high miR-150 and miR-30a-5p baseline levels had lower disposition index (P=0.047 and P=0.007, respectively). The higher risk of disease was associated with high levels (T3) of miR-150 and miR-30a-5p (HR_T3-T1 = 4.218 and HR_T3-T1=2.527, respectively) and low levels (T1) of miR-15a and miR-375 (HR_T1-T3 = 3.269 and HR_T1-T3=1.604, respectively). In conclusion, our study showed that deregulated plasma levels of miR-150, miR-30a-5p, miR-15a, and miR-375 were observed years before the onset of T2DM and pre-DM and could be used to evaluate the risk of developing the disease, which may improve prediction and prevention among individuals at high risk for T2DM.

Emerging biomarkers, tools, and treatments for diabetic polyneuropathy
^{Bönhof GJ, Herder C, Strom A, Papanas N, Roden M, Ziegler D. Endocr Rev. 2019; 40(1): 153–192}

Diabetic neuropathy, with its major clinical sequels, notably neuropathic pain, foot ulcers, and autonomic dysfunction, is associated with substantial morbidity, increased risk of mortality, and reduced quality of life. Despite its major clinical impact, diabetic neuropathy remains underdiagnosed and undertreated. Moreover, the evidence supporting a benefit for causal treatment is weak at least in patients with type 2 diabetes, and current pharmacotherapy is largely limited to symptomatic treatment options. Thus, a better understanding of the underlying pathophysiology is mandatory for translation into new diagnostic and treatment approaches. Improved knowledge about pathogenic pathways implicated in the development of diabetic neuropathy could lead to novel diagnostic techniques that have the potential of improving the early detection of neuropathy in diabetes and prediabetes to eventually embark on new treatment strategies. In this review, we first provide an overview on the current clinical aspects and illustrate the pathogenetic concepts of (pre)diabetic neuropathy. We then describe the biomarkers emerging from these concepts and novel diagnostic tools and appraise their utility in the early detection and prediction of predominantly distal sensorimotor polyneuropathy. Finally, we discuss the evidence for and limitations of the current and novel therapy options with particular emphasis on lifestyle modification and pathogenesis-derived treatment approaches. Altogether, recent years have brought forth a multitude of emerging biomarkers reflecting different pathogenic pathways such as oxidative stress and inflammation and diagnostic tools for an early detection and prediction of (pre)diabetic neuropathy. Ultimately, these insights should culminate in improving our therapeutic armamentarium against this common and debilitating or even life-threatening condition.