Pediatr Nephrol (2015) 30:123–130 DOI 10.1007/s00467-014-2905-5
ORIGINAL ARTICLE
The diagnosis of febrile urinary tract infection in children may be facilitated by urinary biomarkers Hahn-Ey Lee & Do Kyun Kim & Hee Kyung Kang & Kwanjin Park
Received: 10 February 2014 / Revised: 30 June 2014 / Accepted: 2 July 2014 / Published online: 16 August 2014 # IPNA 2014
Abstract Background We prospectively assessed the feasibility of two urinary markers of renal injury as potential diagnostic tests for acute febrile urinary tract infection (UTI) and subsequent renal scarring. Methods The patient cohort comprised children aged 0 to 4 years who visited the emergency room. The children were divided into three groups, namely, a febrile UTI (fUTI), febrile control (FC) and a non-febrile control (NFC) group, respectively, which were matched for sex and age. An enzymelinked immunosorbent assay for neutrophil gelatinaseassociated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) was performed on urine collected from each child. The urine levels of both markers were compared between the three groups, and the diagnostic accuracy was determined based on the area under the receiver-operator characteristic curve (AUC). In the fUTI group, the predictability of subsequent renal scarring was assessed by associating urinary levels with dimercaptosuccinic acid findings 6 months after an UTI episode. H.5 white blood cells (WBCs) detected in a high-power field (HPF)] based on microscopic urinalysis or abnormal urine dipstick findings (positive for leukocyte esterase or nitrite). These combined urine tests have been reported to have 99.8 % sensitivity and 70 % specificity [6]. The diagnosis was confirmed by a positive bacterial urine culture result [a single microorganism ≥105 colony-forming units (CFU)/ml]. Patients with negative urine culture results were excluded from analysis for the strict inclusion of “true” fUTI. Patients with a known history of fUTI, history of antibiotic usage within 72 h, or a history of renal abnormality that might hamper the diagnosis of renal scarring (ureteropelvic junction obstruction, ureteral duplication, horseshoe kidney, etc.) were also excluded. In addition, patients with neutropenia (neutrophils≤0.5×109/L) were excluded. Once an index fUTI patient was enrolled, other febrile patients without evidence of UTI [febrile control (FC)] and non-febrile controls (NFC) were enrolled that matched for age (within 6 months of range) and sex. The patients classified into the FC group had fever with a definite fever focus other than the urinary tract and no laboratory abnormalities except for mild pyuria, which may be associated with viral infections or nonspecific inflammation proximal to the bladder. Patients with-low grade mild pyuria (5–19 WBCs per HPF) were included when these patients showed definite sources of fever in other systems and no positive results in the dipstick analysis, such as nitrate and esterase; these patients were later confirmed not to have fUTI by negative urine culture results. The study was approved by the institutional review board at Seoul National University Hospital. Clinical and laboratory examinations After confirmation of a body temperature of >38.5 °C, blood was collected for plasma CRP (p-CRP) measurement, and
Pediatr Nephrol (2015) 30:123–130
urine was collected for urine examination. The urine from febrile patients was collected by urethral catheterization and that of the NFC patients was collected from a collection bag. The following examinations were performed: dipstick analysis, microscopic examination, culture and enzyme-linked immunosorbent assay (ELISA) for NGAL and KIM-1. We collected data on leukocyte esterase and nitrites from dipsticks used for urinalysis (Multisticks® 7; Siemens, Munich, Germany) and analyzed on a Clinitek Status automated analyzer (Bayer Health Care, Leverkusen, Germany). The results were analyzed dichotomously as ‘negative’ if reported as negative or trace, or as ‘positive’ if 1+ or greater. Data on pyuria were obtained in the microscopic urinalysis. Pyuria was defined as >5 WBCs per HPF in the centrifuged sediment. If a given patient was presumed to have fUTI, admission to hospital was recommended for the administration of parenteral antibiotics, usually lasting 3–5 days until normalization of fever. A technetium Tc 99 m dimercaptosuccinic acid (DMSA) renal scan was checked during admission and any cortical abnormality identified at that time was defined as an acute photon defect. Follow-up DMSA was performed 6 months after admission. Persistence of previous acute photon defects was assumed to indicate permanent renal scarring. ELISA for urinary NGAL and KIM-1 For the determination of urinary NGAL and KIM-1 levels by the ELISA, urine was centrifuged within 1 h of sampling, and aliquots of the supernatants were stored at−70 °C until analysis. Urine samples were thawed approximately 1 h before the assays were performed. For all measurements, 100 ul of diluted urine sample was analyzed in duplicate. For quantification, an 8-point standard curve was prepared by 1:3 dilution of a premixed standard containing all three analytes of a specific panel. The recommended 500-fold dilution was optimal for the detection of NGAL, whereas no dilution was required for the detection of KIM-1. Urinary antigens were bound to the wells of microtiter plates by the incubation of 100-μl urine samples for 1 h at 37 °C. Wells were blocked with buffers containing 5 % bovine serum albumin. The primary antibodies were goat polyclonal antibodies raised against human NGAL (BioPorto Diagnostics, Gentofte, Denmark) and human KIM-1 (USCN Life Science, Hankou, Wuhan, China). Incubation was followed by treatment with horseradish peroxidase-conjugated, affinity-purified rabbit anti-goat immunoglobulin G antibody. 3,3′,5,5′Tetramethylbenzidine substrate (BD™ Biosciences, Franklin Lakes, NJ) was added for color development, which was read after 30 min at 450 nm with a Benchmark Plus® microplate reader (Bio-Rad, Hercules, CA). The urinary creatinine (cr) concentration was used to normalize NGAL and KIM-1 measurements to account for the influence of urinary dilution on its
Pediatr Nephrol (2015) 30:123–130
concentration. Urinary levels of biomarkers were expressed as the respective NGAL/cr or KIM-1/cr (ng/mg cr) ratio. Interassay and intra-assay coefficient variations ranged from 5 to 10 % for batched samples analyzed on the same day. Statistical analysis All variables were expressed as mean±standard deviation . Nonparametric statistics were chosen for analysis due to the relatively small size of the patient population of this study. Statistical analysis was performed using a nonparametric Kruskal–Wallis test for comparison of variables between three groups. To determine whether there was any association between conventional urinary indices of UTI (nitrite, esterase, and pyuria) and each urinary biomarker, we compared means of the urinary biomarkers according to the positivity of each index using Mann–Whitney U tests. A Spearman correlation analysis was performed to determine whether there was any association between the levels of each marker and those of systemic markers of inflammation, i.e. p-CRP and plasma WBC (p-WBC). Receiver operator characteristic (ROC) curves were plotted to compare the performance of the urinary markers, whereby the area under the curve (AUC) served as a measure for the overall ability to discriminate fUTI from the other groups of patients. These curves were also used to determine whether these markers would predict acute photon defects and subsequent renal scarring. The sensitivity and specificity at the best predictive cut-off were calculated for urinary NGAL/cr and KIM-1/cr when the AUC was maximal. The tests were considered significant when 2-tailed p values were 105 CFU/ml) was noted in the urine cultures of all
125
patients in the fUTI group. Patients in the fUTI group showed a higher percentage of increased p-WBC counts and tested positive for nitrite, esterase, and pyuria. In the fUTI group, acute photon defects were observed on the DMSA scans in 18 (55 %) patients. Vesicoureteral reflux was observed in 14 (42 %) of patients in subsequent voiding cystourethrography, and no grade 5 reflux was observed. Comparison of urinary NGAL/cr and KIM-1/cr levels between groups The urinary NGAL/cr or KIM-1/cr ratios in the patients of the two control groups were significantly lower than those in the fUTI patients (Fig. 1). Comparison of these values between the FC and NC patients revealed no significant difference. Association of conventional UTI markers and systemic inflammation Elevated levels of both urinary markers were associated with positive findings of conventional markers of UTI) (Table 2). Comparison of the mean urinary levels of both markers revealed higher levels of both markers in patients who tested positive for urine esterase and pyuria. A correlation analysis between the levels of urinary markers with conventional markers of systemic inflammation showed that both the NGAL/cr and KIM-1/cr ratios were significantly correlated with p-CRP (Spearman’s rho 0.348 for NGAL/cr and 0.441 for KIM-1/cr; p
ORIGINAL ARTICLE
The diagnosis of febrile urinary tract infection in children may be facilitated by urinary biomarkers Hahn-Ey Lee & Do Kyun Kim & Hee Kyung Kang & Kwanjin Park
Received: 10 February 2014 / Revised: 30 June 2014 / Accepted: 2 July 2014 / Published online: 16 August 2014 # IPNA 2014
Abstract Background We prospectively assessed the feasibility of two urinary markers of renal injury as potential diagnostic tests for acute febrile urinary tract infection (UTI) and subsequent renal scarring. Methods The patient cohort comprised children aged 0 to 4 years who visited the emergency room. The children were divided into three groups, namely, a febrile UTI (fUTI), febrile control (FC) and a non-febrile control (NFC) group, respectively, which were matched for sex and age. An enzymelinked immunosorbent assay for neutrophil gelatinaseassociated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) was performed on urine collected from each child. The urine levels of both markers were compared between the three groups, and the diagnostic accuracy was determined based on the area under the receiver-operator characteristic curve (AUC). In the fUTI group, the predictability of subsequent renal scarring was assessed by associating urinary levels with dimercaptosuccinic acid findings 6 months after an UTI episode. H.5 white blood cells (WBCs) detected in a high-power field (HPF)] based on microscopic urinalysis or abnormal urine dipstick findings (positive for leukocyte esterase or nitrite). These combined urine tests have been reported to have 99.8 % sensitivity and 70 % specificity [6]. The diagnosis was confirmed by a positive bacterial urine culture result [a single microorganism ≥105 colony-forming units (CFU)/ml]. Patients with negative urine culture results were excluded from analysis for the strict inclusion of “true” fUTI. Patients with a known history of fUTI, history of antibiotic usage within 72 h, or a history of renal abnormality that might hamper the diagnosis of renal scarring (ureteropelvic junction obstruction, ureteral duplication, horseshoe kidney, etc.) were also excluded. In addition, patients with neutropenia (neutrophils≤0.5×109/L) were excluded. Once an index fUTI patient was enrolled, other febrile patients without evidence of UTI [febrile control (FC)] and non-febrile controls (NFC) were enrolled that matched for age (within 6 months of range) and sex. The patients classified into the FC group had fever with a definite fever focus other than the urinary tract and no laboratory abnormalities except for mild pyuria, which may be associated with viral infections or nonspecific inflammation proximal to the bladder. Patients with-low grade mild pyuria (5–19 WBCs per HPF) were included when these patients showed definite sources of fever in other systems and no positive results in the dipstick analysis, such as nitrate and esterase; these patients were later confirmed not to have fUTI by negative urine culture results. The study was approved by the institutional review board at Seoul National University Hospital. Clinical and laboratory examinations After confirmation of a body temperature of >38.5 °C, blood was collected for plasma CRP (p-CRP) measurement, and
Pediatr Nephrol (2015) 30:123–130
urine was collected for urine examination. The urine from febrile patients was collected by urethral catheterization and that of the NFC patients was collected from a collection bag. The following examinations were performed: dipstick analysis, microscopic examination, culture and enzyme-linked immunosorbent assay (ELISA) for NGAL and KIM-1. We collected data on leukocyte esterase and nitrites from dipsticks used for urinalysis (Multisticks® 7; Siemens, Munich, Germany) and analyzed on a Clinitek Status automated analyzer (Bayer Health Care, Leverkusen, Germany). The results were analyzed dichotomously as ‘negative’ if reported as negative or trace, or as ‘positive’ if 1+ or greater. Data on pyuria were obtained in the microscopic urinalysis. Pyuria was defined as >5 WBCs per HPF in the centrifuged sediment. If a given patient was presumed to have fUTI, admission to hospital was recommended for the administration of parenteral antibiotics, usually lasting 3–5 days until normalization of fever. A technetium Tc 99 m dimercaptosuccinic acid (DMSA) renal scan was checked during admission and any cortical abnormality identified at that time was defined as an acute photon defect. Follow-up DMSA was performed 6 months after admission. Persistence of previous acute photon defects was assumed to indicate permanent renal scarring. ELISA for urinary NGAL and KIM-1 For the determination of urinary NGAL and KIM-1 levels by the ELISA, urine was centrifuged within 1 h of sampling, and aliquots of the supernatants were stored at−70 °C until analysis. Urine samples were thawed approximately 1 h before the assays were performed. For all measurements, 100 ul of diluted urine sample was analyzed in duplicate. For quantification, an 8-point standard curve was prepared by 1:3 dilution of a premixed standard containing all three analytes of a specific panel. The recommended 500-fold dilution was optimal for the detection of NGAL, whereas no dilution was required for the detection of KIM-1. Urinary antigens were bound to the wells of microtiter plates by the incubation of 100-μl urine samples for 1 h at 37 °C. Wells were blocked with buffers containing 5 % bovine serum albumin. The primary antibodies were goat polyclonal antibodies raised against human NGAL (BioPorto Diagnostics, Gentofte, Denmark) and human KIM-1 (USCN Life Science, Hankou, Wuhan, China). Incubation was followed by treatment with horseradish peroxidase-conjugated, affinity-purified rabbit anti-goat immunoglobulin G antibody. 3,3′,5,5′Tetramethylbenzidine substrate (BD™ Biosciences, Franklin Lakes, NJ) was added for color development, which was read after 30 min at 450 nm with a Benchmark Plus® microplate reader (Bio-Rad, Hercules, CA). The urinary creatinine (cr) concentration was used to normalize NGAL and KIM-1 measurements to account for the influence of urinary dilution on its
Pediatr Nephrol (2015) 30:123–130
concentration. Urinary levels of biomarkers were expressed as the respective NGAL/cr or KIM-1/cr (ng/mg cr) ratio. Interassay and intra-assay coefficient variations ranged from 5 to 10 % for batched samples analyzed on the same day. Statistical analysis All variables were expressed as mean±standard deviation . Nonparametric statistics were chosen for analysis due to the relatively small size of the patient population of this study. Statistical analysis was performed using a nonparametric Kruskal–Wallis test for comparison of variables between three groups. To determine whether there was any association between conventional urinary indices of UTI (nitrite, esterase, and pyuria) and each urinary biomarker, we compared means of the urinary biomarkers according to the positivity of each index using Mann–Whitney U tests. A Spearman correlation analysis was performed to determine whether there was any association between the levels of each marker and those of systemic markers of inflammation, i.e. p-CRP and plasma WBC (p-WBC). Receiver operator characteristic (ROC) curves were plotted to compare the performance of the urinary markers, whereby the area under the curve (AUC) served as a measure for the overall ability to discriminate fUTI from the other groups of patients. These curves were also used to determine whether these markers would predict acute photon defects and subsequent renal scarring. The sensitivity and specificity at the best predictive cut-off were calculated for urinary NGAL/cr and KIM-1/cr when the AUC was maximal. The tests were considered significant when 2-tailed p values were 105 CFU/ml) was noted in the urine cultures of all
125
patients in the fUTI group. Patients in the fUTI group showed a higher percentage of increased p-WBC counts and tested positive for nitrite, esterase, and pyuria. In the fUTI group, acute photon defects were observed on the DMSA scans in 18 (55 %) patients. Vesicoureteral reflux was observed in 14 (42 %) of patients in subsequent voiding cystourethrography, and no grade 5 reflux was observed. Comparison of urinary NGAL/cr and KIM-1/cr levels between groups The urinary NGAL/cr or KIM-1/cr ratios in the patients of the two control groups were significantly lower than those in the fUTI patients (Fig. 1). Comparison of these values between the FC and NC patients revealed no significant difference. Association of conventional UTI markers and systemic inflammation Elevated levels of both urinary markers were associated with positive findings of conventional markers of UTI) (Table 2). Comparison of the mean urinary levels of both markers revealed higher levels of both markers in patients who tested positive for urine esterase and pyuria. A correlation analysis between the levels of urinary markers with conventional markers of systemic inflammation showed that both the NGAL/cr and KIM-1/cr ratios were significantly correlated with p-CRP (Spearman’s rho 0.348 for NGAL/cr and 0.441 for KIM-1/cr; p
