A metabolic readout of the urine metabolome of COVID-19 patients.

Analysis of urine samples from COVID-19 patients by 1H NMR reveals important metabolic alterations due to SAR-CoV-2 infection. Previous studies have identified biomarkers in urine that reflect metabolic alterations in COVID-19 patients. We have used 1H NMR to better define these metabolic alterations since this technique allows us to obtain a broad profile of the metabolites present in urine. This technique offers the advantage that sample preparation is very simple and gives us very complete information on the metabolites present. To detect these alterations, we have compared urine samples from COVID-19 patients (n = 35) with healthy people (n = 18). We used unsupervised (Robust PCA) and supervised (PLS-LDA) multivariate analysis methods to evaluate the differences between the two groups: COVID-19 and healthy controls. The differences focus on a group of metabolites related to energy metabolism (glucose, ketone bodies, glycine, creatinine, and citrate) and other processes related to bacterial flora (TMAO and formic acid) and detoxification (hippuric acid). The alterations in the urinary metabolome shown in this work indicate that SARS-CoV-2 causes a metabolic change from a normal situation of glucose consumption towards a gluconeogenic situation and possible insulin resistance.

Role of Urinary Kidney Stress Biomarkers for Early Recognition of Subclinical Acute Kidney Injury in Critically Ill COVID-19 Patients.

A high proportion of critically ill patients with COVID-19 develop acute kidney injury (AKI) and die. The early recognition of subclinical AKI could contribute to AKI prevention. Therefore, this study was aimed at exploring the role of the urinary biomarkers NGAL and [TIMP-2] × [IGFBP7] for the early detection of AKI in this population. This prospective, longitudinal cohort study included critically ill COVID-19 patients without AKI at study entry. Urine samples were collected on admission to critical care areas for determination of NGAL and [TIMP-2] × [IGFBP7] concentrations. The demographic information, comorbidities, clinical, and laboratory data were recorded. The study outcomes were the development of AKI and mortality during hospitalization. Of the 51 individuals that were studied, 25 developed AKI during hospitalization (49%). Of those, 12 had persistent AKI (23.5%). The risk factors for AKI were male gender (HR = 7.57, 95% CI: 1.28-44.8; p = 0.026) and [TIMP-2] × [IGFBP7] ≥ 0.2 (ng/mL)2/1000 (HR = 7.23, 95% CI: 0.99-52.4; p = 0.050). Mortality during hospitalization was significantly higher in the group with AKI than in the group without AKI (p = 0.004). Persistent AKI was a risk factor for mortality (HR = 7.42, 95% CI: 1.04-53.04; p = 0.046). AKI was frequent in critically ill COVID-19 patients. The combination of [TIMP-2] × [IGFBP7] together with clinical information, were useful for the identification of subclinical AKI in critically ill COVID-19 patients. The role of additional biomarkers and their possible combinations for detection of AKI in ritically ill COVID-19 patients remains to be explored in large clinical trials.

Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19.

The utility of the urinary proteome in infectious diseases remains unclear. Here, we analyzed the proteome and metabolome of urine and serum samples from patients with COVID-19 and healthy controls. Our data show that urinary proteins effectively classify COVID-19 by severity. We detect 197 cytokines and their receptors in urine, but only 124 in serum using TMT-based proteomics. The decrease in urinary ESCRT complex proteins correlates with active SARS-CoV-2 replication. The downregulation of urinary CXCL14 in severe COVID-19 cases positively correlates with blood lymphocyte counts. Integrative multiomics analysis suggests that innate immune activation and inflammation triggered renal injuries in patients with COVID-19. COVID-19-associated modulation of the urinary proteome offers unique insights into the pathogenesis of this disease. This study demonstrates the added value of including the urinary proteome in a suite of multiomics analytes in evaluating the immune pathobiology and clinical course of COVID-19 and, potentially, other infectious diseases.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Immune response pattern across the asymptomatic, symptomatic and convalescent periods of COVID-19.

We present an integrated analysis of urine and serum proteomics and clinical measurements in asymptomatic, mild/moderate, severe and convalescent cases of COVID-19. We identify the pattern of immune response during COVID-19 infection. The immune response is activated in asymptomatic infection, but is dysregulated in mild and severe COVID-19 patients. Our data suggest that the turning point depends on the function of myeloid cells and neutrophils. In addition, immune defects persist into the recovery stage, until 12 months after diagnosis. Moreover, disorders of cholesterol metabolism span the entire progression of the disease, starting from asymptomatic infection and lasting to recovery. Our data suggest that prolonged dysregulation of the immune response and cholesterol metabolism might be the pivotal causative agent of other potential sequelae. Our study provides a comprehensive understanding of COVID-19 immunopathogenesis, which is instructive for the development of early intervention strategies to ameliorate complex disease sequelae.

Pilot Study: Long-Term Shedding of SARS-CoV-2 in Urine: A Threat for Dispersal in Wastewater.

Only 4 months after the beginning of SARS-CoV-2 epidemic, the world is facing a global pandemic due to a complex and insidious virus that today constantly poses new challenges. In this study, we highlight a persistent shedding of SARS-CoV-2 RNA into the urine, even in patients with a negative nasopharyngeal swab and in patients considered recovered. What does it mean? Besides the fact that the kidney is a probable site of viral replication, the prolonged viral excretion is a matter of great concern for our drainage system contamination.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

BACKGROUND: AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. METHODS: The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. RESULTS: A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. CONCLUSION: Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Urine biomarkers for the prediction of mortality in COVID-19 hospitalized patients.

Risk factors associated with severity and mortality attributable to COVID-19 have been reported in different cohorts, highlighting the occurrence of acute kidney injury (AKI) in 25% of them. Among other, SARS-CoV-2 targets renal tubular cells and can cause acute renal damage. The aim of the present study was to evaluate the usefulness of urinary parameters in predicting intensive care unit (ICU) admission, mortality and development of AKI in hospitalized patients with COVID-19. Retrospective observational study, in a tertiary care hospital, between March 1st and April 19th, 2020. We recruited adult patients admitted consecutively and positive for SARS-CoV-2. Urinary and serum biomarkers were correlated with clinical outcomes (AKI, ICU admission, hospital discharge and in-hospital mortality) and evaluated using a logistic regression model and ROC curves. A total of 199 COVID-19 hospitalized patients were included. In AKI, the logistic regression model with a highest area under the curve (AUC) was reached by the combination of urine blood and previous chronic kidney disease, with an AUC of 0.676 (95%CI 0.512-0.840; p = 0.023); urine specific weight, sodium and albumin in serum, with an AUC of 0.837 (95% CI 0.766-0.909; p < 0.001) for ICU admission; and age, urine blood and lactate dehydrogenase levels in serum, with an AUC of 0.923 (95%CI 0.866-0.979; p < 0.001) for mortality prediction. For hospitalized patients with COVID-19, renal involvement and early alterations of urinary and serum parameters are useful as prognostic factors of AKI, the need for ICU admission and death.

Urinary cytokines correlate with acute kidney injury in critically ill COVID-19 patients.

BACKGROUND: Acute kidney injury is common in COVID-19 patients admitted to the ICU. Urinary biomarkers are a non-invasive way of assaying renal damage, and so far, urinary cytokines are not fully investigated. The current study aimed to assess urinary cytokine levels in COVID-19 patients. METHODS: Urine was collected from COVID-19 patients (n = 29) in intensive care and compared to a preoperative group of patients (n = 9) with no critical illness. 92 urinary cytokines were analyzed in multiplex using the Olink Target 96 inflammation panel and compared to clinical characteristics, and urinary markers of kidney injury. RESULTS: There were strong correlations between proinflammatory cytokines and between urinary cytokines and urinary kidney injury markers in 29 COVID-19 patients. Several cytokines were correlated to kidney injury, 31 cytokines to AKI stage and 19 cytokines correlated to maximal creatinine. CONCLUSIONS: Urinary inflammatory cytokines from a wide range of immune cell lineages were significantly upregulated during COVID-19 and the upregulation correlated with acute kidney injury as well as urinary markers of kidney tissue damage.

Is coronavirus disease 2019 associated with indicators of long-term bladder dysfunction?

OBJECTIVE: Early reports have suggested that coronavirus disease 2019 (COVID-19) can present with significant urinary frequency and nocturia, and that these symptoms correlate with markers of inflammation in the urine. We evaluated surrogate markers of chronic urinary symptoms to determine if they were more frequent after COVID-19 infection. METHODS: Routinely collected data from the province of Ontario was used to conduct a matched, retrospective cohort study. We identified patients 66 years of age or older who had a positive COVID-19 test between February and May 2020 and survived at least 2 months after their diagnosis. We matched them to two similar patients who did not have a positive COVID-19 test during the same time period. We measured the frequency of urology consultation, cystoscopy, and new prescriptions for overactive bladder medications during a subsequent 3-month period. Proportional hazard models were adjusted for any baseline differences between the groups. RESULTS: We matched 5617 patients with COVID-19 to 11,225 people who did not have COVID-19. The groups were similar, aside from a higher proportion of patients having hypertension and diabetes in the CoVID-19 cohort. There was no significantly increased hazard of new receipt of overactive bladder medication (hazards ratio [HR]: 1.04, p = 0.88), urology consultation (HR: 1.40, p = 0.10), or cystoscopy (HR: 1.14, p = 0.50) among patients who had COVID-19, compared to the matched cohort. CONCLUSION: Surrogate markers of potential bladder dysfunction were not significantly increased in the 2-5 months after COVID-19 infection.

Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis.

Testing is one of the commendable measures for curbing the spread of coronavirus disease (COVID-19). But, it should be done using the most appropriate specimen and an accurate diagnostic test such as real-time reverse transcription-polymerase chain reaction (qRT-PCR). Therefore, a systematic review was conducted to determine the positive detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different clinical specimens using qRT-PCR. A total of 8136 pooled clinical specimens were analyzed to detect SARS-CoV-2, the majority were nasopharyngeal swabs (69.6%). A lower respiratory tract (LRT) specimens had a positive rate (PR) of 71.3% (95% confidence interval [CI]: 60.3%-82.3%) while no virus was detected in the urinogenital specimens. Bronchoalveolar lavage fluid (BLF) specimen had the PR of 91.8% (95% CI: 79.9%-103.7%), followed by rectal swabs; 87.8% (95% CI: 78.6%-96.9%) then sputum; 68.1% (95% CI: 56.9%-79.4%). A low PR was observed in oropharyngeal swabs; 7.6% (95% CI: 5.7%-9.6%) and blood samples; 1.0% (95% CI: -0.1%-2.1%) whereas no SARS-CoV-2 was detected in urine samples. Feces had a PR of 32.8% (95% CI:1 5.8%-49.8%). Nasopharyngeal swab, a widely used specimen had a PR of 45.5% (95% CI: 31.2%-59.7%). In this study, SARS-CoV-2 was highly detected in LRT specimens while no virus was detected in urinogenital specimens. BLF had the highest PR followed by rectal swab then sputum. Nasopharyngeal swab which is widely used had moderate PR. Low PR was recorded in oropharyngeal swab and blood samples while no virus was found in urine samples. Last, the virus was detected in feces, suggesting SARS-CoV-2 transmission by the fecal route.

Discrimination of SARS-CoV-2 infected patient samples by detection dogs: A proof of concept study.

While the world awaits a widely available COVID-19 vaccine, availability of testing is limited in many regions and can be further compounded by shortages of reagents, prolonged processing time and delayed results. One approach to rapid testing is to leverage the volatile organic compound (VOC) signature of SARS-CoV-2 infection. Detection dogs, a biological sensor of VOCs, were utilized to investigate whether SARS-CoV-2 positive urine and saliva patient samples had a unique odor signature. The virus was inactivated in all training samples with either detergent or heat treatment. Using detergent-inactivated urine samples, dogs were initially trained to find samples collected from hospitalized patients confirmed with SARS-CoV-2 infection, while ignoring samples collected from controls. Dogs were then tested on their ability to spontaneously recognize heat-treated urine samples as well as heat-treated saliva from hospitalized SARS-CoV-2 positive patients. Dogs successfully discriminated between infected and uninfected urine samples, regardless of the inactivation protocol, as well as heat-treated saliva samples. Generalization to novel samples was limited, particularly after intensive training with a restricted sample set. A unique odor associated with SARS-CoV-2 infection present in human urine as well as saliva, provides impetus for the development of odor-based screening, either by electronic, chemical, or biological sensing methods. The use of dogs for screening in an operational setting will require training with a large number of novel SARS-CoV-2 positive and confirmed negative samples.

Sequential Analysis of Viral Load in a Neonate and Her Mother Infected With Severe Acute Respiratory Syndrome Coronavirus 2.

We report changes in viral load over time in a 27-day-old neonate with coronavirus disease 2019 who presented with fever, cough, and vomiting. Severe acute respiratory syndrome coronavirus 2 RNA was detected in the nasopharynx, oropharynx, stool, saliva, plasma, and urine. The highest viral RNA copies in nasopharynx decreased over time while viral load in stool remained high.

Proteinuria in COVID-19: prevalence, characterization and prognostic role.

BACKGROUND: Proteinuria has been commonly reported in patients with COVID-19. However, only dipstick tests have been frequently used thus far. Here, the quantification and characterization of proteinuria were investigated and their association with mortality was assessed. METHODS: This retrospective, observational, single center study included 153 patients, hospitalized with COVID-19 between March 28th and April 30th, 2020, in whom total proteinuria and urinary α1-microglobulin (a marker of tubular injury) were measured. Association with mortality was evaluated, with a follow-up until May 7th, 2020. RESULTS: According to the Kidney Disease Improving Global Outcomes staging, 14% (n = 21) of the patients had category 1 proteinuria (< 150 mg/g of urine creatinine), 42% (n = 64) had category 2 (between 150 and 500 mg/g) and 44% (n = 68) had category 3 proteinuria (over 500 mg/g). Urine α1-microglobulin concentration was higher than 15 mg/g in 89% of patients. After a median follow-up of 27 [14;30] days, the mortality rate reached 18%. Total proteinuria and urinary α1-microglobulin were associated with mortality in unadjusted and adjusted models. This association was stronger in subgroups of patients with normal renal function and without a urinary catheter. CONCLUSIONS: Proteinuria is frequent in patients with COVID-19. Its characterization suggests a tubular origin, with increased urinary α1-microglobulin. Tubular proteinuria was associated with mortality in COVID-19 in our restropective, observational study.

Urine abnormalities predict acute kidney injury in COVID-19 patients: An analysis of 110 cases in Chennai, South India.

BACKGROUND AND AIMS: Renal involvement in Covid-19 infection is varied and can affect glomeruli, tubules, interstitium and can cause acute kidney injury (AKI). AKI is a strong predictor of mortality. Routine urinalysis gives an insight into the renal pathology of the patient. We studied the incidence of urinary abnormalities in hospitalised Covid-19 patients and analysed their impact on development of AKI and mortality. METHODS: Information on 110 hospitalised patients with confirmed Covid-19 was retrospectively collected and analysed. The demographic data such as age, gender, comorbid conditions such as diabetes mellitus, the need for dialysis and laboratory data such as urine for albumin, glucose, RBC and WBC, and serum creatinine were collected. The diagnosis of AKI was based on the KDIGO criteria. The outcomes studied were development of AKI and hospital mortality. RESULTS: Urine abnormalities were seen in 71% of the patients. Proteinuria in 58.2%, haematuria in 17.3%, pyuria in 8.2% of patients and concurrent proteinuria and haematuria was seen in 13.6% of patients. AKI was seen in 28.2% of patients and hospital mortality was 24.5%. AKI was strongly associated with mortality. Proteinuria and haematuria were good predictors of development of AKI, more strongly when they occurred concurrently (p < 0.01). CONCLUSION: Our results suggest that urine analysis is a simple test, which can be used to predict development of AKI and mortality and may be used for risk stratification of Covid-19 patients, especially in low resource settings.

COVID-19 patients in intensive care develop predominantly oliguric acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is a syndrome of reduced glomerular filtration rate and/or reduced urine flow associated with mortality in corona virus disease 2019 (COVID-19). AKI is often associated with renal tissue damage, which may lead to chronic kidney disease. Biomarkers of tissue damage may identify patients of particular risk. METHODS: In a prospective observational study of 57 patients admitted to intensive care, AKI incidence and characteristics was evaluated according to KDIGO criteria and related to days after admission. Urinary albumin, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule 1 (KIM-1) and Plasma Tissue Inhibitor of MetalloProteinase 2 (TIMP-2) were analysed in 52 patients at admission. The majority (n = 51, 89%) of patients developed AKI, and 27 (47%) patients had predominantly oliguric AKI where oliguria was more severe than plasma Creatinine increase. Severe oliguria within first 2 days after admission was common (n = 37, 65%), whereas stage 2 and 3 AKI due to Creatinine occurred later than day 2 in 67% (12/18) of cases. Renal replacement therapy was started in 9 (16%) patients, and 30-day mortality was 28%. Urinary biomarkers were increased in a majority of patients, but did not robustly predict KDIGO stage. Most patients had microalbuminuria, and severe albuminuria (albumin Creatinine ratio > 30 mg/mmol) was found in n = 9 (17%) patients. CONCLUSIONS: A majority of patients with COVID-19 admitted to the ICU develop AKI. The functional deficit is often low urinary volume, and initial levels of biomarkers are generally increased without clear relation to final AKI stage.

A simultaneous exploratory and quantitative amino acid and biogenic amine metabolic profiling platform for rapid disease phenotyping via UPLC-QToF-MS.

Metabolic phenotyping using mass spectrometry (MS) is being applied to ever increasing sample numbers in clinical and epidemiology studies. High-throughput and robust methods are being developed for the accurate measurement of metabolites associated with disease. Traditionally, quantitative assays have utilized triple quadrupole (QQQ) MS based methods; however, the use of such focused methods removes the ability to perform discovery-based metabolic phenotyping. An integrated workflow for the hybrid simultaneous quantification of 34 biogenic amines in combination with full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping is presented. Primary and secondary amines are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate prior to revered-phase liquid chromatographic separation and mass spectrometric detection. Using the HRAM-MS data, retrospective phenotypic data mining could be performed, demonstrating the versatility of HRAM-MS instrumentation in a clinical and molecular epidemiological environment. Quantitative performance was assessed using two MS detector platforms: Waters TQ-XS (QQQ; n = 3) and Bruker Impact II QToF (HRAMS-MS; n = 2) and three human biofluids (plasma, serum and urine). Finally, each platform was assessed using a certified external reference sample (NIST SRM 1950 plasma). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1-400 µmol L-1. Quantitative values were comparable across all instruments for human plasma, serum and urine samples, and calculated concentrations were verified against certified reference values for NIST SRM 1950 plasma as an external reference. As a real-life biological exemplar, the method was applied to plasma samples obtained from SARS-CoV-2 positive patients versus healthy controls. Both the QQQ and QToF approaches were equivalent in being able to correctly classify SARS-CoV-2 positivity. Critically, the use of HRAM full scan data was also assessed for retrospective exploratory mining of data to extract additional biogenic amines of biomarker interest beyond the 34 quantified targets.

Association of Proteinuria and Hematuria with Acute Kidney Injury and Mortality in Hospitalized Patients with COVID-19.

INTRODUCTION: Acute kidney injury (AKI) is strongly associated with poor outcomes in hospitalized patients with coronavirus disease 2019 (COVID-19), but data on the association of proteinuria and hematuria are limited to non-US populations. In addition, admission and in-hospital measures for kidney abnormalities have not been studied separately. METHODS: This retrospective cohort study aimed to analyze these associations in 321 patients sequentially admitted between March 7, 2020 and April 1, 2020 at Stony Brook University Medical Center, New York. We investigated the association of proteinuria, hematuria, and AKI with outcomes of inflammation, intensive care unit (ICU) admission, invasive mechanical ventilation (IMV), and in-hospital death. We used ANOVA, t test, χ2 test, and Fisher's exact test for bivariate analyses and logistic regression for multivariable analysis. RESULTS: Three hundred patients met the inclusion criteria for the study cohort. Multivariable analysis demonstrated that admission proteinuria was significantly associated with risk of in-hospital AKI (OR 4.71, 95% CI 1.28-17.38), while admission hematuria was associated with ICU admission (OR 4.56, 95% CI 1.12-18.64), IMV (OR 8.79, 95% CI 2.08-37.00), and death (OR 18.03, 95% CI 2.84-114.57). During hospitalization, de novo proteinuria was significantly associated with increased risk of death (OR 8.94, 95% CI 1.19-114.4, p = 0.04). In-hospital AKI increased (OR 27.14, 95% CI 4.44-240.17) while recovery from in-hospital AKI decreased the risk of death (OR 0.001, 95% CI 0.001-0.06). CONCLUSION: Proteinuria and hematuria both at the time of admission and during hospitalization are associated with adverse clinical outcomes in hospitalized patients with COVID-19.