Abstract Diabetes mellitus (DM) is a major cause of chronic kidney disease (CKD) and changes in lipoprotein. Literature data suggest that cholesterol associated with high-density lipoprotein (HDLc) plays a protective role against albuminuria and diabetic nephropathy. These factors are related with changes in serum apolipoproteins such Apo A-I. This work aims to evaluate the correlation of apolipoprotein A-I with renal function in diabetic outpatients. Samples were collected from 281 outpatients for analyses of glycated hemoglobin (HbA1c), serum creatinine (SCr), urea (BUN), cholesterol (TC), triglycerides (TG), HDL-cholesterol (HDLc) and estimation of glomerular filtration rate (eGFR). Urinary samples were obtained to assess urine albumin creatinine ratio (UACR). Triglyceride-glucose index (TyG) was calculated to estimate insulin resistance. The mean age was 61,5 ± 16,4 years. Most patients were female, Afro-Mestizo and over 50 years of age. Pearson's revealed negative correlation of creatinine with HDLc and Apo A-I. There was a correlation of eGFR with HbA1c, TG and HDLc. TyG presented a negative correlation with Apo A-I and HDLc. The highest quartiles of UACR presented the highest TyG and the lowest Apo A-I. In conclusion, Apo A-I indicated an association with insulin resistance and changes in renal function parameters, especially as a factor associated with the onset of albuminuria, with a better predictive potential than the HDLc.

ABSTRACT Introduction: Supplementation with probiotics for patients with chronic kidney disease (CKD) may be associated with decreased systemic inflammation. Objective: To assess the impact of oral supplementation with probiotics for patients with CKD on hemodialysis. Method: This double-blind randomized clinical trial included 70 patients on hemodialysis; 32 were given oral supplementation with probiotics and 38 were in the placebo group. Blood samples were collected at the start of the study and patients were given oral supplementation with probiotics or placebo for three months. The probiotic supplement comprised four strains of encapsulated Gram-positive bacteria: Lactobacillus Plantarum A87, Lactobacillus rhamnosus, Bifidobacterium bifidum A218 and Bifidobacterium longum A101. Patients were given one capsule per day for 3 months. Blood samples were taken throughout the study to check for inflammatory biomarkers. Non-traditional biomarkers Syndecan-1, IFN-y, NGAL, and cystatin C were measured using an ELISA kit, along with biochemical parameters CRP, calcium, phosphorus, potassium, PTH, GPT, hematocrit, hemoglobin, glucose, and urea. Results: Patients given supplementation with probiotics had significant decreases in serum levels of syndecan-1 (239 ± 113 to 184 ± 106 ng/mL, p = 0.005); blood glucose levels also decreased significantly (162 ± 112 to 146 ± 74 mg/dL, p = 0.02). Conclusion: Administration of probiotics to patients with advanced CKD was associated with decreases in syndecan-1 and blood glucose levels, indicating potential improvements in metabolism and decreased systemic inflammation.

Resumo Introdução: A suplementação com probióticos na doença renal crônica (DRC) pode estar associada à redução do processo inflamatório sistêmico. Objetivo: Avaliar a suplementação oral com probióticos em pacientes com DRC em hemodiálise. Método: Ensaio clínico, duplo cego, randomizado com 70 pacientes em hemodiálise, sendo 32 do grupo que recebeu o suplemento de probióticos e 38 do grupo placebo. Inicialmente ocorreu a coleta de sangue e suplementação oral com probióticos ou placebo durante três meses. O suplemento probiótico foi composto pela combinação de 4 cepas de bactérias Gram-positivas encapsuladas: Lactobacillus Plantarum A87, Lactobacillus rhamnosus, Bifidobacterium bifidum A218 e Bifidobacterium longum A101, sendo 1 cápsula do suplemento ao dia, durante 3 meses. Após esse período foram feitas novas coletas de sangue para dosagem dos biomarcadores inflamatórios. Foram analisados os biomarcadores não tradicionais: Syndecan-1, IFN-y, NGAL e cistatina C pelo método ELISA, e os seguintes parâmetros bioquímicos: PCR, cálcio, fósforo, potássio, PTH, TGP, hematócrito, hemoglobina, glicose e ureia. Resultados: Os pacientes que receberam suplemento tiveram diminuição significativa dos níveis séricos de syndecan-1 (de 239 ± 113 para 184 ± 106 ng/mL, p = 0,005). Outro parâmetro que diminuiu significativamente nos pacientes que receberam suplemento foi a glicemia (de 162 ± 112 para 146 ± 74 mg/dL, p = 0,02). Conclusão: O uso de probióticos na DRC avançada esteve associado à redução dos níveis de syndecan-1 e glicemia, sinalizando possível melhora no metabolismo e redução do processo inflamatório sistêmico.

Abstract Ischemia/reperfusion (I/R) -induced Acute kidney injury (AKI) is characterized by hypoxia and production of reactive oxygen species (ROS), which could be prevented with antioxidant agents. The essential oil of Lippia alba (EOLA) chemotype citral-limonene is rich in components with antioxidant activity. So, this study aims to evaluate the nephroprotective effect of the EOLA on in vivo and in vitro models of renal I/R. Male Wistar rats were submitted to right nephrectomy, followed by ischemia by clamping the renal artery in the left kidney and a reperfusion. Animals received EOLA (200 mg/kg) or vehicle three days prior to I/R surgery. Blood and urine samples were obtained to evaluate creatinine, urea, uric acid, and creatinine clearance. The left kidney was collected for histological evaluations and analysis of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH). HK-2 cells were used to evaluate the effect of EOLA on I/R in vitro, using the MTT assay. Moreover, scanning electron microscopy (SEM) was performed to evaluate cell morphological alterations. The I/R resulted in biochemical parameter alterations, with EOLA reverting all these alterations. Histological evaluation showed that EOLA protected the morphological changes caused by I/R. Also, EOLA was able to reduce TBARS and increase GSH levels in renal tissue. In the tubular cells, the EOLA partially reversed the damage caused by I/R, which was observed through the SEM. The EOLA showed effect against I/R-induced AKI through pre-treatment, protecting biochemical and oxidative parameters in vivo and reversing tubular cell damage in vitro.

ABSTRACT Background: The long-term effects of schistosomiasis on the glomerulus may contribute to the development of chronic kidney disease. This study aimed to investigate baseline Schistosoma mansoni-Circulating Anodic Antigen (CAA) levels and their association with kidney biomarkers related to podocyte injury and inflammation in long-term follow-up after praziquantel (PZQ) treatment. Methods: Schistosoma infection was diagnosed by detecting CAA in urine using a quantitative assay based on lateral flow using luminescent up-converting phosphor reporter particles. A cutoff threshold of 0.1 pg/mL CAA was used to diagnose Schistosoma infection (baseline) in a low-prevalence area in Ceará, Northeast, Brazil. Two groups were included: CAA-positive and CAA-negative individuals, both of which received a single dose of PZQ at baseline. Urinary samples from 55 individuals were evaluated before (baseline) and at 1, 2, and 3 years after PZQ treatment. At all time points, kidney biomarkers were quantified in urine and adjusted for urinary creatinine levels. Results: CAA-positive patients had increased baseline albuminuria and proteinuria and showed greater associations between kidney biomarkers. CAA levels correlated only with Vascular Endothelial Growth Factor (VEGF) (podocyte injury) levels. Increasing trends were observed for malondialdehyde (oxidative stress), monocyte chemoattractant protein-1 (inflammation marker), and VEGF. In the follow-up analysis, no relevant differences were observed in kidney biomarkers between the groups and different periods. Conclusions: S. mansoni-infected individuals presented subclinical signs of glomerular damage that may reflect podocyte injury. However, no causal effect on long-term renal function was observed after PZQ treatment.

Abstract Ischemia/reperfusion injury (I/R) is commonly related to acute kidney injury (AKI) and oxidative stress. Antioxidant agents are used to treat this condition. Lippia sidoides is a brazillian shrub with anti-inflammatory and anti-oxidative properties. Thus, the aim of this study is to evaluate the effect of Lippia sidoides ethanolic extract (LSEE) on in vivo and in vitro models of AKI induced by I/R. Male Wistar rats were submitted to unilateral nephrectomy and ischemia on contralateral kidney for 60 min via clamping followed by reperfusion for 48 h. They were divided into four groups: Sham, LSEE (sham-operated rats pre-treated with LSEE), I/R (rats submitted to ischemia) and I/R-LSEE (rats treated with LSEE before ischemia). Kidney tissues homogenates were used to determine stress parameters and nephrin expression. Plasma and urine samples were collected for biochemical analysis. I/R in vitro assays were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and flow cytometry assays in Rhesus Monkey Kidney Epithelial Cells (LLC-MK2). The LSEE treatment prevented biochemical and nephrin expression alterations, as well as oxidative stress parameters. In the in vitro assay, LSEE protected against cell death, reduced the reactive oxygen species and increased mitochondrial transmembrane potential. LSEE showed biotechnological potential for a new phytomedicine as a nephroprotective agent.

Abstract Changes in lipoprotein metabolism are among the main causes of hemodynamic impairment in renal function. COVID-19 is an multisystemic inflammatory disease, aggravating this situation. This cross-sectional study investigated the relationship of serum lipoprotein profile with inflammatory parameters and renal function in 95 COVID-19 outpatients in comparison with 173 with flu-like symptoms. Serum samples were collected for the determination of total cholesterol and fractions, apolipoproteins (Apo A-I and Apo B), urea (sUr) and creatinine (sCr). The glomerular filtration rate (eGFR) was calculated. Neutrophil/lymphocyte (NLR) and platelet/lymphocyte (PLR) ratios were calculated as inflammatory parameters derived from the blood tests. COVID-19 patients presented lower high-density lipoprotein cholesterol (HDL-c) (47.90 ± 1.543 vs. 51.40 ± 0.992) and higher PLR (190.9 ± 9.410 vs. 137.6 ± 5.534) and NLR (3.40 ± 0.22 vs. 2.80 ± 0.15). Both NLR and PLR correlated with each other (r = 0.639). Furthermore, the Apo B/Apo A-I ratio was correlated with PLR (r = 0.5818) and eGFR (r = -0.2630). COVID-19 patients classified as at high risk of developing acute myocardial infarction based on the Apo B/ Apo A-I ratio had higher values for sUr/sCr. Thus, serum apolipoproteins, PLR, and NLR could be related to renal dysfunction in COVID-19.

Resumo A lesão renal aguda (LRA) é comum na Unidade de Terapia Intensiva Neonatal (nUTI) e a sepse é uma de suas principais causas, especialmente em prematuros. Apresenta altas taxas de mortalidade e sua detecção precoce é o primeiro passo para a prevenção dessa condição, pois permite o tratamento adequado e melhora o desfecho, diminui o tempo de internação, os custos não médicos e a morbimortalidade. Destaca-se ainda que a LRA pode evoluir para doença renal crônica (DRC), havendo a necessidade de diálise, com maior risco de desenvolver doenças cardiovasculares. Este artigo de revisão tem como objetivo discutir a LRA em recém-nascidos (RNs) prematuros com sepse, abordando biomarcadores utilizados na rotina laboratorial e principalmente a utilização de biomarcadores não tradicionais para identificação precoce de LRA.

Abstract Acute kidney injury (AKI) is a common finding in Neotatal Intensive Care Units (NICU). Sepsis is one the main causes of AKI in preterm newborns. AKI has been associated with significant death rates. Early detection of the condition is the first step to improving prevention, treatment, and outcomes, while decreasing length of hospitalization, care costs, and morbimortality. AKI may progress to chronic kidney disease (CKD), a condition linked with dialysis and greater risk of cardiovascular disease. This review article aims to discuss cases of AKI in preterm newborns with sepsis, the use of biomarkers in lab workup, and the use of non-conventional biomarkers for the early identification of AKI.

Resumo Introdução: A suplementação com probióticos na doença renal crônica (DRC) pode estar associada à redução do processo inflamatório sistêmico. Objetivo: Avaliar a suplementação oral com probióticos em pacientes com DRC em hemodiálise. Método: Ensaio clínico, duplo cego, randomizado com 70 pacientes em hemodiálise, sendo 32 do grupo que recebeu o suplemento de probióticos e 38 do grupo placebo. Inicialmente ocorreu a coleta de sangue e suplementação oral com probióticos ou placebo durante três meses. O suplemento probiótico foi composto pela combinação de 4 cepas de bactérias Gram-positivas encapsuladas: Lactobacillus Plantarum A87, Lactobacillus rhamnosus, Bifidobacterium bifidum A218 e Bifidobacterium longum A101, sendo 1 cápsula do suplemento ao dia, durante 3 meses. Após esse período foram feitas novas coletas de sangue para dosagem dos biomarcadores inflamatórios. Foram analisados os biomarcadores não tradicionais: Syndecan-1, IFN-y, NGAL e cistatina C pelo método ELISA, e os seguintes parâmetros bioquímicos: PCR, cálcio, fósforo, potássio, PTH, TGP, hematócrito, hemoglobina, glicose e ureia. Resultados: Os pacientes que receberam suplemento tiveram diminuição significativa dos níveis séricos de syndecan-1 (de 239 ± 113 para 184 ± 106 ng/mL, p = 0,005). Outro parâmetro que diminuiu significativamente nos pacientes que receberam suplemento foi a glicemia (de 162 ± 112 para 146 ± 74 mg/dL, p = 0,02). Conclusão: O uso de probióticos na DRC avançada esteve associado à redução dos níveis de syndecan-1 e glicemia, sinalizando possível melhora no metabolismo e redução do processo inflamatório sistêmico.

ABSTRACT Introduction: Supplementation with probiotics for patients with chronic kidney disease (CKD) may be associated with decreased systemic inflammation. Objective: To assess the impact of oral supplementation with probiotics for patients with CKD on hemodialysis. Method: This double-blind randomized clinical trial included 70 patients on hemodialysis; 32 were given oral supplementation with probiotics and 38 were in the placebo group. Blood samples were collected at the start of the study and patients were given oral supplementation with probiotics or placebo for three months. The probiotic supplement comprised four strains of encapsulated Gram-positive bacteria: Lactobacillus Plantarum A87, Lactobacillus rhamnosus, Bifidobacterium bifidum A218 and Bifidobacterium longum A101. Patients were given one capsule per day for 3 months. Blood samples were taken throughout the study to check for inflammatory biomarkers. Non-traditional biomarkers Syndecan-1, IFN-y, NGAL, and cystatin C were measured using an ELISA kit, along with biochemical parameters CRP, calcium, phosphorus, potassium, PTH, GPT, hematocrit, hemoglobin, glucose, and urea. Results: Patients given supplementation with probiotics had significant decreases in serum levels of syndecan-1 (239 ± 113 to 184 ± 106 ng/mL, p = 0.005); blood glucose levels also decreased significantly (162 ± 112 to 146 ± 74 mg/dL, p = 0.02). Conclusion: Administration of probiotics to patients with advanced CKD was associated with decreases in syndecan-1 and blood glucose levels, indicating potential improvements in metabolism and decreased systemic inflammation.

Abstract INTRODUCTION: This study estimated the seroprevalence and risk factors of Chagas disease (CD) in a population of the Quixeré municipality, Ceará. METHODS: We conducted serological methods to detect the Trypanosoma cruzi infection. The other variables were evaluated by a standardized questionnaire. RESULTS: The estimated prevalence of CD was 3.7%. Male sex, age >40 years, being farmers, low education level, origin from rural areas, and being born in Quixeré were significantly associated with infection. CONCLUSION: CD persists in this rural population of Northeast Brazil. Poverty, low education, and limited information regarding CD are critical issues that need to be addressed.

Resumo COVID-19 é uma pandemia associada a manifestações clínicas sistêmicas. Neste estudo, apresenta-se revisão narrativa acerca do envolvimento renal na COVID-19. Envolvimento renal parece ser relacionado a efeitos citopáticos diretos, mecanismos imunológicos, efeitos indiretos de outros mediadores no tecido renal, além de disfunção e lesão de outros órgãos. A evolução da COVID-19 pode ser complicada por lesão renal aguda (LRA) em percentual significativo dos pacientes, e a disfunção renal parece ser associada a pior prognóstico. Pacientes com doença renal crônica (DRC) parecem ser mais suscetíveis a formas severas da COVID-19. Pacientes em terapia de substituição renal (TSR) contínua também constituem população vulnerável em razão de idade avançada, comorbidades subjacentes, resposta imune disfuncional e aglomeração em unidades de diálise, com necessidade de visitas frequentes aos serviços de saúde. Pacientes transplantados renais podem estar em alto risco dadas imunossupressão a longo prazo e comorbidades; assim, o manejo da imunossupressão é mandatório. Finalmente, TSR pode ser necessária durante a COVID-19, e diferentes modalidades são discutidas conforme manifestações clínicas e aspectos laboratoriais. Assim, COVID-19 parece acometer os rins por diferentes mecanismos, os quais contribuem para o desenvolvimento de LRA e aumento da severidade da doença. Ainda, pacientes com DRC e transplantados renais apresentam elevado risco para desenvolvimento de COVID-19 e de mortalidade.

Abstract COVID-19 is a pandemic associated with systemic clinical manifestations. In this study, we aimed to present a narrative review on kidney involvement in COVID-19. Kidney involvement could be derived from direct cytopathic effects, immunological mechanisms, indirect effects on renal tissue through other mediators, and dysfunction or injury of other organs. The evolution of COVID-19 may be complicated with acute kidney injury (AKI) in a significant percentage of patients, and renal dysfunction seems to be associated with worse prognosis. Patients with chronic kidney disease (CKD) seem to be more susceptible to the severe forms of COVID-19. Patients with renal replacement therapy (RRT) are also a vulnerable population as consequence of their advanced age, underlying comorbidities, impaired immune response, and clustering in hemodialysis centers, with requirements for frequent contact with healthcare services. Kidney transplant patients may be at high-risk due to long-term immunosuppression and comorbidities, hence, managing immunosuppression is imperative. Lastly, renal replacement therapy may be required during COVID-19, and different modalities are discussed based on clinical findings and laboratorial aspects. Therefore, COVID-19 seems to affect kidney by different mechanisms, which contributes for AKI development and increases the severity of the disease. Also, patients with CKD and kidney transplant recipients are at higher risk for COVID-19 and mortality.

Abstract Ischemia-reperfusion (I/R) plays an important role in the process of acute kidney injury (AKI) due to the generation of reactive oxygen species (ROS). Substances of natural origin have been studied in the prevention of oxidative damage related to I/R. Quercetin is a flavonoid with antioxidant potential and modulate enzymes, such the inhibition of the Rennin-Angiotensin System (RAS). The aim of this study is to evaluate the nephroprotective effect of quercetin against the I/R and analyze the inhibition of RAS. Rhesus monkey Kidney Epithelial Cells (LLC-MK2 line) were submitted to an in vitro ischemia/reperfusion model. After the reperfusion cells were treated with quercetin, the cell viability was accessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Tubular cell damage was assessed by the Kidney Injury Molecule-1 (KIM-1) measurement. Oxidative stress was evaluated through Thiobarbituric Acid Reactive Substances (TBARS) and reduced glutathione (GSH). The evaluation of cell death and the mitochondrial depolarization were analyzed by flow cytometry. Quercetin prevents cell death reducing oxidative stress and preventing mitochondrial membrane depolarization. Molecular docking showed that quercetin prevents cell damage better than losartan and lisinopril, inhibitors of RAS. Quercetin has a potential to interact with type 1 angiotensin II receptor (AT1) with greater affinity through the formation of five hydrogen bonds of strong intensity.

ABSTRACT Leprosy may present kidney and endothelial abnormalities, being a risk factor for complications. However, the involvement of renal and vascular endothelia has been poorly investigated. We aimed to investigate if the levels of systemic endothelial biomarkers are associated with kidney abnormalities and the clinical forms of leprosy. This is a cross-sectional study with leprosy patients enrolled in January 2017 to December 2018, before the initiation of the multidrug therapy. Leprosy-associated clinical and epidemiological data were collected. Two groups were investigated: Paucibacillary (PB) and Multibacillary (MB) infections, for the comparisons. Serum and urine samples were obtained for laboratory analysis. In serum samples, were evaluated the endothelial biomarkers VCAM-1 and ICAM-1. In total, 101 leprosy patients were included, the mean age was 48±١٥ years and 71 (70%) were male. The multibacillary form occurred in 81 cases (80%), among which 22 had the Virchowian form. Serum creatinine was more elevated in the MB group than in PB patients. In addition, VCAM-1 was elevated in the MB group and was correlated with the bacteriological index (rho = 0.372, p <0.01), the duration of disease symptoms (rho = 0.234, p = 0.04), and the number of skin lesions (rho = 0.468, p <0.001). Moreover, in MB patients who presented albuminuria >15 mg/g of creatinine, VCAM-1 showed a significant correlation with increased albuminuria and improved the correlation with the number of skin lesions (rho= 0.563, p=0.010). In conclusion, higher systemic VCAM-1 levels were associated with the multibacillary clinical form of leprosy and with increased albuminuria. Prospective studies are necessary to establish a cause-effect and evaluate the preventive role of these biomarkers to improve the clinical care.

Abstract Bothrops are one of the most common medically important snakes found in Latin America. Its venom is predominantly hemotoxic and proteolytic, which means that local lesion (edema and redness) and hemorrhagic symptoms are recurrent in envenoming by this snake. Although hemorrhage is usually the major cause of death, snakebite-related acute kidney injury is another potentially fatal clinical complication that may lead to chronic kidney disease. The present review highlights the main studies on Bothrops venom-related acute kidney injury, including observational, cross-sectional, case-control and cohort human studies available up to December 2019. The following descriptors were used according to Medical Subject Headings (MeSH): on Medline/Pubmed and Google Scholar “acute kidney injury” or “kidney disease” and “Bothrops”; on Lilacs and SciELO “kidney disease” or “acute kidney injury” and “Bothrops”. Newcastle-Ottawa quality assessment scale was used to appraise the quality of the cross-sectional and cohort studies included. The selection of more severe patients who looked for health care units and tertiary centers is a risk of bias. Due to the methodological heterogeneity of the studies, a critical analysis of the results was performed based on the hypothesis that the design of the included studies influences the incidence of acute kidney injury. Fifteen human studies (total participants 4624) were included according to stablished criteria. The coagulation abnormalities (hemorrhagic symptoms, abnormal fibrinogen and activated partial thromboplastin time) were associated with acute kidney injury in the most recent studies reported. The findings observed in this review provide up-to-date evidence about the acute kidney injury pathogenesis following Bothrops syndrome. Studies pointed out that coagulation abnormalities comprise the major pathway for acute kidney injury development. This review may improve patient management by primary healthcare providers, allowing earlier diagnosis and treatment of Bothrops venom-related acute kidney injury.

ABSTRACT Tropical diseases are mainly found in the tropical regions of Asia, Africa and Latin America. They are a major Public Health problem in these regions, most of them are considered neglected diseases and remain as important contributors to the development of AKI (Acute Kidney Injury), which is associated with increased patients’ morbidity and mortality. In most countries, kidney disease associated to tropical diseases is attended at health services with poor infrastructure and inadequate preventive measures. The long-term impacts of these infections on kidney tissue may be a main cause of future kidney disease in these patients. Therefore, the investigation of novel kidney injury biomarkers in these tropical diseases is of utmost importance to explain the mechanisms of kidney injury, to improve their diagnosis and prognosis, as well as the assessment to health systems by these patients. Since 2011, our group has been studying renal biomarkers in visceral and cutaneous leishmaniasis, schistosomiasis, leptospirosis and leprosy. This study has increased the knowledge on the pathophysiology of kidney disease in the presence of these infections and has contributed to the early diagnosis of kidney injury, pointing to glomerular, endothelial and inflammatory involvement as the main causes of the mechanisms leading to nephropathy and clinical complications. Future perspectives comprise establishing long-term cohort groups to assess the development of kidney disease and the patients’ survival, as well as the use of new biomarkers such as urinary exosomes to detect risk groups and to understand the progression of kidney injuries.

Abstract INTRODUCTION: Loxoscelism is a clinical condition involving spiders of the genus Loxosceles. One of the most severe complications is acute kidney injury (AKI). This study aimed to investigate AKI and other complications associated with loxoscelism. METHODS: We analyzed cases diagnosed with loxoscelism in an area where most accidents were caused by Loxosceles amazonica from January 2010 to December 2015. AKI was defined according to the KDIGO criteria. RESULTS: Forty-five patients were recorded: 95.6% presented characteristic necrotic skin lesions and 13.3% AKI. CONCLUSIONS: Loxoscelism could cause kidney involvement which is uncommon and could lead to the death of these patients.