Acta Pzdiatr Scand 64: 182-186, 1975
USE OF T H E NITROBLUE TETRAZOLIUM (NBT) TEST IN T H E DIFFERENTIATION BETWEEN PY ELON EPHRI TIS AND CYSTITIS B. BJORKSTEN and P. de C H A T E A U From the Department of Paediatrics, Umed Universitv, U m e d , Sweden
ABSTRACT. Bjorksten, B. and de Chateau, P. (Department of Paediatrics, Umefi University, Umefi, Sweden.) Use of the Nitroblue Tetrazolium (NBT) Test in the differentiation between pyelonephritis and cystitis. Acta Paediatr Scand 64: 182, 1975.NBT tests were performed on blood from 37 patients with urinary tract infections caused by coliform bacteria. The level of infections was evaluated by the clinical signs, ESR, renal concentrating capacity and titre of antibodies against the strains of E . coli isolated in their urine. Elevated proportions of NBT-positive neutrophils were found in 11 of 14 patients with pyelonephritis and in 3 of 23 patients with cystitis or asymptomatic bacteriuria (ABU). The total number of NBT-positive neutrophils was 1000 or more per mm” blood in 11 of 13 patients considered to have pyelonephritis, while it was 800 or less in all the patients investigated, evaluated as having cystitis or asymptomatic baderiuria. The NBT test is recommended as an adjunct in the level diagnosis or urinary tract infections in children. The utility of the test in smouldering pyelonephritis is presently being investigated.
KEY WORDS: Nitroblue tetrazolium test, pyelonephritis, cystitis, level diagnosis in urinary tract infection 0
The differentiation of lower urinary tract infections from pyelonephritis in childhood is difficult when based solely on clinical symptoms. In addition to fever, loin pain, elevated ESR, lowered renal conce,ntrating capacity and elevated titres of E . coli antibodies indicate pyelonephritis (2). Since evaluation of the last two parameters is timeconsuming and the methods are not always available, there is an obvious need for other methods for level differentiation. By the Nitroblue Tetrazolium (NBT) Test introduced by Park e t al. in 1968 (14), neutrophilic granulocytes can be divided into NBT-positive and NBT-negative, depending on whether they reduce the dye into nitroblue formazan or not. The test has been recommended as an aid in the differentiation of bacterial, fungal and parasitic infections Acta PiPdiatr Scand 64
from other febrile conditions (1, 8, 15). After standardizing the test method however, we found that it is of limited value in the differentiation of streptococcal and non-streptococcal throat infections (7) and in the diagnosis of perforated appendicitis (8). The aim of this study was to investigate whether the NBT test could be used as an aid in the differentiation of pyelonephritis from urinary tract infections not involving the renal parenchyma. MATERIAL All patients admitted to the study had at least one bacterial count of 1OOOOO coliform microorganisms or more per ml of urine. The patients with asymptomatic bacteriuria (ABU) had several positive urine cultures ( 6 patients) or had one positive urinary culture and pyuria of at least 170 white cells per mm’ uncentnfugated urine (2 patients). The study included 34 fe-
NBT-test in pyelonephritis and cystitis males and 3 males aged 6 months to 24 years, with an average age of 6 years. The diagnosis with regard to the site of infection was based on measuring the rectal temperature, the micro erythrocyte sedimentation rate (ESR), the renal concentrating capacity and the titre of E . coli antibody. As abnormal values of these items were considered a temperature of 38°C or more, an ESR of at least 20 m m per hour, a renal concentrating capacity of less than 800 mosmlkg water and a fourfold or greater rise in E . coli antibody titre to at least 256. The infec!ion was considered to be a pyelonephritis when at least three of these four items were pathological. When none of the items were abnormal and the clinical symptoms were micturition abnormalities only, the patients were classified as having cystitis. According to these criteria the patients were separated into three groups: Group 1: 8 patients with pyelonephritis Group 2: I I patients with cystitis and 5 patients with
ABU Group 3: 13 patients who could not be classified within group 1 or 2. The records of these 13 patients were scrutinized without knowledge of the results of the NBT tests, and the patients were classified according to their symptoms and laboratory findings in two subgroups: 3 A , 6 patients with probable pyelonephritis and group 3 B, 7 patients with probable cystitis or ABU.
The patients received appropriate chemotherapy, usually sulphonamide for 10 days and were readmitted to the hospital after 2 to 10 weeks for a clinical and laboratory check-up with urine culture, urinary cell count, NBT-test, ESR. renal concentrating capacity and titration of antibodies against E . coli.
* METHODS Urine specimens for culture were saved after thorough cleansing of the external genitalia with soap and water. When possible, a mid-stream portion was used. The urine specimens were immediately chilled to 4°C and kept at this temperature until culture was performed. This was done with a calibrated loop technique and the E . coli-isolates were submitted to simplified 0-group ing (13). The blood leucocytes were counted in capillary blood. The number of neutrophils was obtained by multiplying the total white blood cell count with the percentage of neutrophilic granulocytes, or by counting the number of polymorphonuclear cells in a Burker chamber. The urinary leucocytes were counted in uncentrifuged fresh urine in a Fuchs-Rosenthal chamber. The osmolality of urine was determined after 10-14 hours’ water deprivation. If the osmolality of urine was less than 800 mosm/kg water, the renal concentrating capacity was determined after water deprivation for 16-17 hours and administration of pitressin tannate as described by WinLerg (17). Blood samples for titration of E. coli antibodies wert drawn within a few days after onset of symptoms or, in the asymptomatic patients, when they were admitted to the study. A second blood sample was then
I
4 4
c
r’
m
z *
183
E
n 38
3A
GROUP I
2
Fig. 1. Percentage of NBT-positive neutrophils in 8 patients with pyelonephritis (group 1); 6 with probable pyelonephritis (group 3 A); 7 with probable cystitis or ABU (group 3 B) and 16 with cystitis or ABU (group 2).
drawn when the patients were admitted for the checkup. The titration was performed using the E . coli strain isolated from the patient’s urine as antigen (18). Sera from patients whose bacteria were not 0-grouped were tested for E . co/i antibodies using a pool of eight common E . coli 0-antigens (01, 02, 04, 06, 07, 08, 018, 075) ( 3 ) . In order to evaluate the titre of IgM antibodies against E . coli the sera were treated with mercaptoethanol as described by Holngren ( 10). The NBT tests and endotoxin stimulated NBT tests were performed within 6 hours of drawing the blood samples and as described elsewhere ( 5 ) using venous blood with 10-20 IU heparin (Vitrum AB, Stockholm) added per ml of blood. The samples were incubated with a 0.1% solution of NBT in saline in a 37°C waterbath for 25 minutes and smears were made on glass slides and counterstained with Giemsa stain. One hundred neutrophils were counted and the percentage of neutrophils containing intracellular deposits of forma-
+ z GROUP
1
3A
36
2
Fig. 2. Number of NBT-positive neutrophils expressed in thousands per mm? blood in 8 patients with pyelonephritis (group I ) ; 5 with probable pyelonephritis (group 3 A ) ; 7 with probable cystitis or ABU (group 3 B) and 14 with cystitis or ABU (group 2). Arra Prediatr Scond 64
184
B . Bjorkstgn and P . de Chateau
Table 1. Some clinical data on and laboratory findings in 37 patients with urinary tract infection White cells in blood per mm?
Neutrophils in blood per mmS
I5 600 21 300 23 OOO 10 200 16000 13 500 21 100 10 400
Group 3 A . Probable pyelonephritis (6) 9 9/12 Rec. 10 10 1st 11 6/12 1 st 12 22 8/12 Rec . 13 10 Rec. 14 6/12 1st Group 2 . C-vstitis ( I I ) and ABU ( 5 ) 15 4 6/12 Rec. ABU Rec. ABU 16 6 2/12 17 7 8/12 Rec . 18 24 2/12 Rec. Rec. 19 20 1/12 Rec. ABU 20 1 Rec. 21 12 3/12 22 6 6/12 1st Rec . 23 9 1/12 24 9 4/12 1st. mu 25 5 9/12 1st Rec. 26 10 27 11 7/12 1st 28 9 2/12 Rec. ABU 29 2 2/12 Rec. 30 6 2/12 Rec.
Pat. no.
Type of infection”
Age
Group I . Pyelonephritis I 7 2 7/12 3 loll2 4 8/12 5 10112 6 6/12 7 6/12 8 1 6/12
(8) Rec . 1st 1st 1st
I st 1st Rec. Rec.
%
permm:’
12800 15 900 18 900 6 100 1 1 200 9 500 I6 800 6 300
27 26 27 37 32 41
3500 4100 5100 2400 3600 3900 1700
17600 6 300 12 600 ND 5 100 10 000
8 200 3 200 6 200 ND 2 600 4 600
34 4 29 36 6 30
2800 100 1800 ND 200 1400
6 900 7 200 4 600 6 800 12700 6 400 4 100 5 200 6 900 6 300 ND 5 400 12400 6 800 5 100 3 900
2 600 3 100 2 300 2 700 9 700 1 600 ND I500 4 200 2 200 ND 2 800 10 400 4 100 700 1 200
23 5 5 12 3 6 I 4 1 21 5 8 1
600 200 100 300 300
5 100 10300 3 400 4 100 2 100 2 400 3 300
8 6 3 2 8 32 3
Group 3 B . Probable cystitis ( 4 )and probable ABU (3) 31 8 10112 1st 6 500 32 12 2/12 Rec . 14 100 33 14 8/12 Rec . 6 600 9 1/12 Rec. ABU 7 300 34 35 5 7/12 Rec . 4 000 Rec. ABU 5 500 36 2 5j12 37 4 7/12 Rec. ABU 5 500 a
NBT-positive neutrophils
10 16
1
0 2
1000
100
ND 100 100
500 ND 200 100
100 0 100
400 600 100 100
200 800 100
Type of infection: 1st or recurrent (rec.), asymptomatic bacteriuria (ABU) ND=not done.
zan, “NBT-positive cells”, were recorded. With this method, normally less than 13% of the neutrophils are NBT-positive (7). The total number of NBT-positive neutrophils was obtained by multiplying the number of neutrophils per mm3 blood by the percentage of NBTpositive neutrophils. This number is not normally more than 500 per mm3 blood in healthy children (6). For statistical evaluation of the results the ranking test for unpaired measurements developed by Wilcoxon was used (16). Acta Pediotr Scond
64
RESULTS The NBT test results are given in Figs. 1 and 2 and Table 1. The proportions of NBTpositive neutrophils were elevated in 11 of the 14 patients with a definite or probable pyelonephritis (Table 1, Fig. 1). Of the 23 patients with definite or probable cystitis
NBT-test in pyelonephritis and cystitis or ABU, 3 patients with ABU had an elevated proportion of NBT-positive neutrophils. The total number of NBT-positive neutrophils was at least 1000 cells per mm3 blood in 11 of 13 patients with definite or probable pyelonephritis, while it was 800 or less in all the 21 patients investigated with definite o r probable cystitis o r ABU (Table 1, Fig. 2). The total white blood cell and neutrophil counts were significantly higher @
USE OF T H E NITROBLUE TETRAZOLIUM (NBT) TEST IN T H E DIFFERENTIATION BETWEEN PY ELON EPHRI TIS AND CYSTITIS B. BJORKSTEN and P. de C H A T E A U From the Department of Paediatrics, Umed Universitv, U m e d , Sweden
ABSTRACT. Bjorksten, B. and de Chateau, P. (Department of Paediatrics, Umefi University, Umefi, Sweden.) Use of the Nitroblue Tetrazolium (NBT) Test in the differentiation between pyelonephritis and cystitis. Acta Paediatr Scand 64: 182, 1975.NBT tests were performed on blood from 37 patients with urinary tract infections caused by coliform bacteria. The level of infections was evaluated by the clinical signs, ESR, renal concentrating capacity and titre of antibodies against the strains of E . coli isolated in their urine. Elevated proportions of NBT-positive neutrophils were found in 11 of 14 patients with pyelonephritis and in 3 of 23 patients with cystitis or asymptomatic bacteriuria (ABU). The total number of NBT-positive neutrophils was 1000 or more per mm” blood in 11 of 13 patients considered to have pyelonephritis, while it was 800 or less in all the patients investigated, evaluated as having cystitis or asymptomatic baderiuria. The NBT test is recommended as an adjunct in the level diagnosis or urinary tract infections in children. The utility of the test in smouldering pyelonephritis is presently being investigated.
KEY WORDS: Nitroblue tetrazolium test, pyelonephritis, cystitis, level diagnosis in urinary tract infection 0
The differentiation of lower urinary tract infections from pyelonephritis in childhood is difficult when based solely on clinical symptoms. In addition to fever, loin pain, elevated ESR, lowered renal conce,ntrating capacity and elevated titres of E . coli antibodies indicate pyelonephritis (2). Since evaluation of the last two parameters is timeconsuming and the methods are not always available, there is an obvious need for other methods for level differentiation. By the Nitroblue Tetrazolium (NBT) Test introduced by Park e t al. in 1968 (14), neutrophilic granulocytes can be divided into NBT-positive and NBT-negative, depending on whether they reduce the dye into nitroblue formazan or not. The test has been recommended as an aid in the differentiation of bacterial, fungal and parasitic infections Acta PiPdiatr Scand 64
from other febrile conditions (1, 8, 15). After standardizing the test method however, we found that it is of limited value in the differentiation of streptococcal and non-streptococcal throat infections (7) and in the diagnosis of perforated appendicitis (8). The aim of this study was to investigate whether the NBT test could be used as an aid in the differentiation of pyelonephritis from urinary tract infections not involving the renal parenchyma. MATERIAL All patients admitted to the study had at least one bacterial count of 1OOOOO coliform microorganisms or more per ml of urine. The patients with asymptomatic bacteriuria (ABU) had several positive urine cultures ( 6 patients) or had one positive urinary culture and pyuria of at least 170 white cells per mm’ uncentnfugated urine (2 patients). The study included 34 fe-
NBT-test in pyelonephritis and cystitis males and 3 males aged 6 months to 24 years, with an average age of 6 years. The diagnosis with regard to the site of infection was based on measuring the rectal temperature, the micro erythrocyte sedimentation rate (ESR), the renal concentrating capacity and the titre of E . coli antibody. As abnormal values of these items were considered a temperature of 38°C or more, an ESR of at least 20 m m per hour, a renal concentrating capacity of less than 800 mosmlkg water and a fourfold or greater rise in E . coli antibody titre to at least 256. The infec!ion was considered to be a pyelonephritis when at least three of these four items were pathological. When none of the items were abnormal and the clinical symptoms were micturition abnormalities only, the patients were classified as having cystitis. According to these criteria the patients were separated into three groups: Group 1: 8 patients with pyelonephritis Group 2: I I patients with cystitis and 5 patients with
ABU Group 3: 13 patients who could not be classified within group 1 or 2. The records of these 13 patients were scrutinized without knowledge of the results of the NBT tests, and the patients were classified according to their symptoms and laboratory findings in two subgroups: 3 A , 6 patients with probable pyelonephritis and group 3 B, 7 patients with probable cystitis or ABU.
The patients received appropriate chemotherapy, usually sulphonamide for 10 days and were readmitted to the hospital after 2 to 10 weeks for a clinical and laboratory check-up with urine culture, urinary cell count, NBT-test, ESR. renal concentrating capacity and titration of antibodies against E . coli.
* METHODS Urine specimens for culture were saved after thorough cleansing of the external genitalia with soap and water. When possible, a mid-stream portion was used. The urine specimens were immediately chilled to 4°C and kept at this temperature until culture was performed. This was done with a calibrated loop technique and the E . coli-isolates were submitted to simplified 0-group ing (13). The blood leucocytes were counted in capillary blood. The number of neutrophils was obtained by multiplying the total white blood cell count with the percentage of neutrophilic granulocytes, or by counting the number of polymorphonuclear cells in a Burker chamber. The urinary leucocytes were counted in uncentrifuged fresh urine in a Fuchs-Rosenthal chamber. The osmolality of urine was determined after 10-14 hours’ water deprivation. If the osmolality of urine was less than 800 mosm/kg water, the renal concentrating capacity was determined after water deprivation for 16-17 hours and administration of pitressin tannate as described by WinLerg (17). Blood samples for titration of E. coli antibodies wert drawn within a few days after onset of symptoms or, in the asymptomatic patients, when they were admitted to the study. A second blood sample was then
I
4 4
c
r’
m
z *
183
E
n 38
3A
GROUP I
2
Fig. 1. Percentage of NBT-positive neutrophils in 8 patients with pyelonephritis (group 1); 6 with probable pyelonephritis (group 3 A); 7 with probable cystitis or ABU (group 3 B) and 16 with cystitis or ABU (group 2).
drawn when the patients were admitted for the checkup. The titration was performed using the E . coli strain isolated from the patient’s urine as antigen (18). Sera from patients whose bacteria were not 0-grouped were tested for E . co/i antibodies using a pool of eight common E . coli 0-antigens (01, 02, 04, 06, 07, 08, 018, 075) ( 3 ) . In order to evaluate the titre of IgM antibodies against E . coli the sera were treated with mercaptoethanol as described by Holngren ( 10). The NBT tests and endotoxin stimulated NBT tests were performed within 6 hours of drawing the blood samples and as described elsewhere ( 5 ) using venous blood with 10-20 IU heparin (Vitrum AB, Stockholm) added per ml of blood. The samples were incubated with a 0.1% solution of NBT in saline in a 37°C waterbath for 25 minutes and smears were made on glass slides and counterstained with Giemsa stain. One hundred neutrophils were counted and the percentage of neutrophils containing intracellular deposits of forma-
+ z GROUP
1
3A
36
2
Fig. 2. Number of NBT-positive neutrophils expressed in thousands per mm? blood in 8 patients with pyelonephritis (group I ) ; 5 with probable pyelonephritis (group 3 A ) ; 7 with probable cystitis or ABU (group 3 B) and 14 with cystitis or ABU (group 2). Arra Prediatr Scond 64
184
B . Bjorkstgn and P . de Chateau
Table 1. Some clinical data on and laboratory findings in 37 patients with urinary tract infection White cells in blood per mm?
Neutrophils in blood per mmS
I5 600 21 300 23 OOO 10 200 16000 13 500 21 100 10 400
Group 3 A . Probable pyelonephritis (6) 9 9/12 Rec. 10 10 1st 11 6/12 1 st 12 22 8/12 Rec . 13 10 Rec. 14 6/12 1st Group 2 . C-vstitis ( I I ) and ABU ( 5 ) 15 4 6/12 Rec. ABU Rec. ABU 16 6 2/12 17 7 8/12 Rec . 18 24 2/12 Rec. Rec. 19 20 1/12 Rec. ABU 20 1 Rec. 21 12 3/12 22 6 6/12 1st Rec . 23 9 1/12 24 9 4/12 1st. mu 25 5 9/12 1st Rec. 26 10 27 11 7/12 1st 28 9 2/12 Rec. ABU 29 2 2/12 Rec. 30 6 2/12 Rec.
Pat. no.
Type of infection”
Age
Group I . Pyelonephritis I 7 2 7/12 3 loll2 4 8/12 5 10112 6 6/12 7 6/12 8 1 6/12
(8) Rec . 1st 1st 1st
I st 1st Rec. Rec.
%
permm:’
12800 15 900 18 900 6 100 1 1 200 9 500 I6 800 6 300
27 26 27 37 32 41
3500 4100 5100 2400 3600 3900 1700
17600 6 300 12 600 ND 5 100 10 000
8 200 3 200 6 200 ND 2 600 4 600
34 4 29 36 6 30
2800 100 1800 ND 200 1400
6 900 7 200 4 600 6 800 12700 6 400 4 100 5 200 6 900 6 300 ND 5 400 12400 6 800 5 100 3 900
2 600 3 100 2 300 2 700 9 700 1 600 ND I500 4 200 2 200 ND 2 800 10 400 4 100 700 1 200
23 5 5 12 3 6 I 4 1 21 5 8 1
600 200 100 300 300
5 100 10300 3 400 4 100 2 100 2 400 3 300
8 6 3 2 8 32 3
Group 3 B . Probable cystitis ( 4 )and probable ABU (3) 31 8 10112 1st 6 500 32 12 2/12 Rec . 14 100 33 14 8/12 Rec . 6 600 9 1/12 Rec. ABU 7 300 34 35 5 7/12 Rec . 4 000 Rec. ABU 5 500 36 2 5j12 37 4 7/12 Rec. ABU 5 500 a
NBT-positive neutrophils
10 16
1
0 2
1000
100
ND 100 100
500 ND 200 100
100 0 100
400 600 100 100
200 800 100
Type of infection: 1st or recurrent (rec.), asymptomatic bacteriuria (ABU) ND=not done.
zan, “NBT-positive cells”, were recorded. With this method, normally less than 13% of the neutrophils are NBT-positive (7). The total number of NBT-positive neutrophils was obtained by multiplying the number of neutrophils per mm3 blood by the percentage of NBTpositive neutrophils. This number is not normally more than 500 per mm3 blood in healthy children (6). For statistical evaluation of the results the ranking test for unpaired measurements developed by Wilcoxon was used (16). Acta Pediotr Scond
64
RESULTS The NBT test results are given in Figs. 1 and 2 and Table 1. The proportions of NBTpositive neutrophils were elevated in 11 of the 14 patients with a definite or probable pyelonephritis (Table 1, Fig. 1). Of the 23 patients with definite or probable cystitis
NBT-test in pyelonephritis and cystitis or ABU, 3 patients with ABU had an elevated proportion of NBT-positive neutrophils. The total number of NBT-positive neutrophils was at least 1000 cells per mm3 blood in 11 of 13 patients with definite or probable pyelonephritis, while it was 800 or less in all the 21 patients investigated with definite o r probable cystitis o r ABU (Table 1, Fig. 2). The total white blood cell and neutrophil counts were significantly higher @
