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Nitroblue tetrazolium dye test as an aid in thedifferential diagnosis of febrile disorders
230
February, 1971 T h e Journal o/ P E D I A T R I C S
Nitroblue tetrazolium dye test as an aid in the differential diagnosis of febrile disorders Nitroblue tetrazolium (NBT) dye tests were performed upon blood obtained at time of admission or during the course of hospitalization from 247 febrile patients and 20 afebrile individuals. Detailed clinical and laboratory diagnostic evaluation permitted subsequent placement of 181 of the febrile patients into 8 specific groups. Knowledge of only percentage and absolute number of NBT-positive cells permitted reclassification into 4 groups as [oUows: (l) normal subjects, (2) individuals with viral infection, noninfectious fevers, or partially treated bacterial infection, (3) untreated bacterial infection, and (4) bacterial infection unresponsive to therapy provided. There was generally no correlation between percentage of NBT-positive cells and either white blood cell counts or body temperature recorded at the time blood [or the NBT test was obtained. Discriminant analysis was used to prepare a nomogram which permits classification of patients prospectively into one of the 4 groups, if the percentage and absolute number of NBT-positive cells are known.
Ralph D. Feigin, M.D., ~ Penelope G. Shackelford, M.D., Sung C. Choi, Ph.D., Kathryn K. Flake, M.D., Frank A. Franklin, Jr., M.D., and Carl S. Eisenberg, M.D. ST.
LOUIS~
i'VfO.
N E U T R O P H I L S from patients with chronic granulomatous disease of childhood have a diminished bactericidal capacity? Studies of the function and metabolism of these leukocytes have shown that although
From the Department of Pediatrics, and the Division of Biostatistics, Washington University School of Medicine, and the Division of Infectious Diseases, St. Louis Children's Hospital. Reprint address: Dr. Feigln, St. Louls Children's Hospital. 500 S. Kingshi~hway, St. Louis, Mo. 63110. ~'Reeipient of United States Public Health Service Research Career Development Award No. 1KO4A146206 from the National Institute o] Allergy and Infectious Diseases. Supported in part by a Public Health Service Training Grant No. AH00068-05.
Vol. 78, No. 2, pp. 230-237
they ingest bacteria normally,2 intracellular killing of organisms is delayed,a During the course of studies of the respiratory response of Ieukocytes to phagocytosis in this disease, it was noted that the cells failed to reduce nitroblue tetrazolium (NBT) dye.4 Baehner /
See Editor's column, p. 376. and Nathan * attributed this defect to a deficiency of nicotinamide adenine dinucleotide reduced (NADH) oxidase and proposed that the NBT test could be used to detect patients with chronic granulomatous disease. A large number of normal peripheral neu-
Volume 78 Number 2
trophils will reduce NBT dye during phagocytosis in vitro, whereas only small numbers of neutrophils reduce this dye spontaneously. During the course of a bacterial infection in a person with normal neutrophil function, the infecting organism undergoes phagocytosis by neutrophils in vivo. Park and associates s proposed that bacterial infection could induce sufficient metabolic changes in these neutrophils to result in spontaneous in vitro reduction of NBT dye by an increased proportion of neutrophils when compared to healthy control subjects or to patients with nonbacterial disease. They tested this postulate and reported that both the percentage and absolute number of NBT-positive cells were significantly increased in bacterial infection when compared to normal individuals or patients with nonbacterial illnesses. Moreover, there was almost no overlap in the percentage or absolute number of NBTpositive cells in patients with bacterial infection when compared with all other groups. These results suggested that this test might be a simple and useful method for differentiating bacterial infection from nonbacterial disease. Although the results of Park and associates s were of great interest, the precise statistical significance of differences between groups was not presented. In addition, the nurnber of patients with bacterial infections, exclusive of those with meningitis, was small (6),, suggesting that if a larger number of these patients were sampled, greater overlap between some patients with bacterial infection and those in other groups might be observed. The potential effect of previous antibiotic therapy upon the result of the NBT dye test in patients with bacterial infection was not assessed, and the rapidity with which the N B T dye test might return to normal following effective therapeutic intervention was not stated. Several other questions remained unanswered including: (1) Is there a positive, negative, or no correlation between total white blood cell count and percentage of NBT-positive cells? (2) Is the NBT dye test superior to a routine white blood cell count in helping to dis-
N B T test in [ebrile disorders
23 1
tinguish between bacterial and nonbacterial diseases? (3) Does the NBT test have a positive, negative, or no correlation with body temperature at the time the test is performed? (4) With what degree of predictability can one categorize the individual febrile patient if the percentage and absolute number of NBT-positive cells is known ? The usefulness of the NBT dye test as a routine diagnostic aid in the evaluation of patients with infection is dependent upon obtaining answers to these and other similar questions. The present study was undertaken in an attempt to provide this information, as well as to extend previous observations to a larger number of individuals. MATERIAL
AND METHODS
Patient selection. Heparinized blood (1 to 2 ml.) was obtained from 247 patients at the time of admission or during the course of their hospitalization for a febrile illness. For the purposes of this study, patients were considered febrile if a temperature of 101 ~ F. or greater, rectally, was sustained for at least 12 hours or a similar temperature elevation was recorded on at least 3 consecutive days. Heparinized blood (1 to 2 ml.) was also obtained from 20 afebrile control patients (group 1) who had been electively admitted for minor surgical procedures. All patients were between 3 months and 15 years of age. Patients admitted with a febrile illness were the recipients of a detailed diagnostic evaluation which permitted the subsequent placement of 181 of these individuals into specific groups. Patients categorized as having bacterial meningitis (group 2) included only those individuals in whom the diagnosis was established by positive culture of pathogenic bacteria from the cerebrospinal fluid. Individuals categorized as "other bacterial infections" (group 3) included only patients with a well-recognized clinical syndrome, i.e., osteomyelitis, septic arthritis, pharyngitis, pneumonia, impetigo, etc., and from whom a pathogenic bacterium was isolated from the site of infection or the blood. Several patients with bacterial meningitis and other
232
Feigln et aI.
untreated bacterial infections were followed with serial NBT dye tests to determine the rapidity with which the number of NBTpositive ceils returned to normal. Patients classified as having a pretreated bacterial infection (group 4) included individuals with similar types of culturally identified meningeal or nonmeningeal infections but who had received antibiotic therapy prior to the performance of the NBT dye test. In addition, the antibiotic therapy was appropriate for the disease as determined by dose, route of administration, and sensitivity testing for the organism isolated. Patients considered to have noninfectious fevers (group 5) were those with a diagnosis of juvenile rheumatoid arthritis, systemic lupus erythematosus, granulomatous colitis, Letterer-Siwe disease, or chemical pneumonitis determined by appropriate clinical and laboratory criteria. In addition, evidence for concurrent bacterial or viral infection was sought but not found. Individuals categorized as having viral meningitis (group 6) included only patients with clinical signs of meningeal irritation, no previous antibiotic therapy, cerebrospinal fluid changes consistent with the diagnosis, and complete resolution of the disease process without antibiotic therapy. Patients classified as having other viral infections (group 7) included only those individuals with a readily recognizable viral syndrome, i.e., mumps, rubeola, rubella, varicella, herpes simplex, etc., or other individuals from whom a specific virus was isolated and deemed to be of clinical significance because of a fourfold or greater rise in antibody titer to that virus. Patients with ineffectively pretreated bacterial infections (group 8) included those with culturally identified bacterial diseases who were receiving an antibiotic which was deemed ineffective by sensitivity testing for the organism isolated. Four febrile patients with fungal diseases were identified, 2 with pulmonary histoplasmosis, 1 with esophageal moniliasis, and 1 with chronic mucocutaneous moniliasis. Recording of data. Special forms were
The Journal o/ Pediatrics February 1971
prepared to facilitate subsequent computer analysis of the data and 43 specific variables recorded for each patient. Information recorded of specific importance with regard to this report included patient's age, clinical diagnosis, cultural or serologic evidence of infection, previous antibiotic therapy including its duration prior to NBT dye test and its efficacy, duration of symptomatology prior to performance of the NBT dye test, body temperature at the time the NBT dye test was performed, percentage and absolute number of NBT-positive cells, and results of white blood cell and differential counts. Laboratory methods. The NBT dye test was performed as reported by Park and associates? Wright-stained smears were examined under the microscope with oil, and 100, 200, or 500 neutrophils were counted. Since evaluation of this method during the first 4 months of this study indicated that separate results were in good agreement, the percentage of NBT-positive cells was subsequently determined on the basis of a 100 cell count. Only those neutrophils with a large black deposit were considered to be NBT positive. At the same time that blood was obtained for the NBT dye test, a total white blood cell count and differential count was performed. The absolute number of nentrophils per cubic millimeter was calculated. The absolute number of NBT-positive neutrophils was determined from the absolute number of neutrophils and the percentage of NBT-positive cells. Performance of this test can be accomplished within 1 to 2 hours of the time that blood is obtained. Statistleal analysis. Statistical analysis was performed by the Division of Biostatistics, Washington University School of Medicine. Possible differences between the 8 groups in terms of percentage of NBT-positive cells as well as absolute number of NBT-positive cells were investigated by the least significant difference test of which the first stage is analysis of variance. Individual group comparisons were investigated as in the second stage of the least significant difference test by the appropriate t test.
Volume 78 Number 2
N B T test in febrile disorders
The potential effect of total white blood cell count and temperature on percentage of NBT-positive cells was studied by computing the correlation coefficients between total white blood cell count and percentage of NBT-positive cells, and temperature and NBT-positive cells for each group. Before analysis, total white blood cell count and temperature were transformed by taking logarithms, and the angular transformation was employed on the percentage of NBTpositive cells to satisfy the statistical assumptions. To determine the degree of predictability with which one can categorize the individual patient if both the percentage and absolute number of NBT-positive cells is known, discriminant analysis was employed. ~ RESULTS
The percentage and absolute number of NBT-positive cells, as well as the standard deviation and ranges for each group, are shown in Table I. It is apparent that both percentage and absolute number of NBTpositive cells are increased in all groups above normal control subjects with the ex-
233
ception of patients with fungal diseases (group 9). Individuals with fungal disease were excluded from further statistical consideration because of their small number. Analysis of variance indicated that there were significant differences between the 8 groups in terms of both percentage of NBT-positive cells (p < 0.001) and absolute number of NBT-positive cells (p < 0.001). On the basis of the percentage and absolute number of NBT-positive cells, all patients tested can be placed into one of 4 larger groups. Afebrile control individuals (group A) differ significantly (p < 0.001) from patients in all other groups and have the lowest mean values. Patients with treated bacterial infection, viral meningitis, other viral infections, and noninfectious fevers do not differ significantly from each other (group B), but significant differences (p < 0.001) are noted when these patients are compared to all other groups. Patients with bacterial meningitis or other bacterial infections (group C) are similar to each other but differ significantly (p < 0.001) from all other groups, whereas individuals with in-
Table I. Results of nitroblue tetrazolium dye test in 181 febrile patients and in 20 control individuals Mean % NBTpositive cells +-ISD 8.75 + 5.25
Ran~.e 2-17
Mean absolute No. I NBT-positive cells +- ISD 420.5 + 278.0
Range 69-949
Group No. 1 Control subjects
No. 20
2 Bacterial meningitis
8
37.1 -+ 16.8
18-70
4331.5 -+ 3237.6
167-9392
3 Other bacterial infections
28
41.3 + 14.9
6-71
3992.7 + 2776.9
784-8760
4 Pretreated bacterial infections
56
22.7 + I8.35
1-60
2358.9 -+ 2 6 1 6 . 4
87-5672
5 Noninfectious fevers
17
14.5 + 13.7
4-42
1706
6 Viral meningitis
24
18.8 + 12.3
2-55
1124.5 -+ 7 4 5 . 4
7 Other viral infections
33
20.2 + 15.6
2-60
1095
-+ 1209.1
8 Pretreated bacterial (ineffective)
11
54.9 + 14.4
31-72
4850
+ 4195
1317-16043
4
8.0 -+ 10.0
2-23
529
-+ 748
130-1650
9 Fungal infections
-+ 2 2 0 2
145-8940 173-2970 34-4516
234
Feigin et al.
The Journal o[ Pediatrics February 1971
effectively treated bacterial infection (group D) differ from all other groups (p < 0.001). T h e correlation coefficients between percentage of NBT-positive cells and either total white bloGd cell count or body temperature are generally small and with one exception not significant at the 5 per cent level based on appropriate degrees of freedom. There is a significant correlation (p < 0.01) between total white blood cell count and percentage of NBT-positive cells in patients with treated bacterial infections. I n all other groups the percentage of N B T positive cells is independent of both the total white blood ceil count and body temperature recorded at the time that blood for the N B T test was obtained. T h e period of time necessary for the percentage of NBT-positive cells to increase above normal was impossible to assess. In some individuals with bacterial meningitis sampled within 12 hours of onset of clinical symptoms, the percentage of NBT-positive cells was already over 30. I n others with bacterial infection and well-documented symptoms for 4 to 5 days, the percentage of NBT-positive cells was similar. T h e rapidity with which the percentage of NBT-positive cells can return to normal is perhaps best illustrated by a 15-monthold patient with Hemophilus influenzae meningitis followed with serial evaluation (Table I I ) . It is apparent that effective antibacterial therapy for as short a time as 18 hours can dramatically reduce the percentage of NBT-positive cells toward normal. Independence of the percentage of NBT-positive cells and the total white blood cell count is also noted. T h e patient's cerebrospinal fluid was sterile after the first 36
hours of therapy. T h e marked elevation in white cell count noted at 114 hours was associated with the development of a subdural effusion which proved to be sterile. The development of this effusion did not affect the percentage of NBT-positive cells which continued to fall to within the normal range. To enhance the clinical applicability of the results obtained, discriminant analysis was employed utilizing the assumption that efficiency of prediction would be greater if one utilized both the percentage and absolute n u m b e r of NBT-positive cells. A discriminant function was obtained representing each of the 4 basic groups of individuals. T h e y are: for group A f1 = -66.8 - 0.71 (% NBT) - 0.019 (Abs. NBT) + 58.5 (log10 Abs. NBT) + 0.00023 (% NBT x Abs. NBT), for group B f, z -76.2 - 0.68 (% NBT) - 0.019 (Abs. NBT) + 61.8 (Iog10 Abs. NBT) + 0.00023 (% NBT x Abs. NBT), for group C f3 = -78.8 - 0.58 (% NBT) - 0.018 (Abs. NBT) + 61.4 (log10 Abs. NBT) + 0.00022 (% NBT x Abs. NBT), and for group D f~ = -84.7 - 0.56 (% NBT) - 0.020 (Abs. NBT) + 62.8 (log~0 Abs. NBT) + 0.00026 (% NBT x Abs. NBT). T h e percentage and absolute n u m b e r of NBT-positive cells obtained for any given individual can be substituted appropriately in each equation. T h e probability that an individual is a m e m b e r of each group can be computed from fl, f2, fa, and f4, and he
Table II. Hemophilus influenzae meningitis
Time o[ treatment Pretreatment After therapy 18 hr. 42 hr. 11~4 hr.
I NBT-positive cells Absolute No. NBT1 (%) positive cells 52 7,972 28 14 8
2,848 1,180 1,066
Total white blood cell count 21,600
% neutrophils 71
18,500 14,800 34,200
55 57 39
Volume 78 Number 2
N B T test in /ebrile disorders
patients is shown in Table I I I . For example, if only the percentage and absolute number of NBT-positive cells were known for the 20 afebrile control individuals (group A), 14 could have been classified correctly while 6 would have been misclassified to group B. None would have been placed in group C or D, groups representing untreated or ineffectively treated bacterial infections, respectively. All misclassified afebrile individuals are o n the border line between group A and Group B when the percentage and absolute number of NBT-positive cells are plotted on the nomogram.
is best classified as a member of the group represented by the largest f value. To facilitate the placement of any given patient into a specific group, a nomogram (Fig. 1) has been prepared based upon the 4 equations which were derived from the data. For any given patient, the point of intersection of the percentage of NBT-positive cells and absolute number of NBT-positive cells permits visual categorization of the patient into one of the 4 groups (A, B, C, D). Probability of correct classification increases as the distance of the point from any dividing line increases. Conversely, if the point of intersection lies near a dividing line, the probability of correct classification decreases. If the point lies precisely on a dividing line, there is an equal probability that the patient should be classified in the groups on either side of the line. The efficiency of the classification procedure when applied retrospectively to our
DISCUSSION Park and associates s have previously reported that the nitroblue tetrazolium dye test might be useful in differentiating patients with bacterial infection from individuals with nonbacterial disease. They showed that the mean proportion of NBT-positive cells was 8.5 per cent in healthy control
I~ooo
• 8,000 x~ 6,000
4,000
k
2.oo0 'K
0 ~
0
", :
-~.~
235
.
-~
~
...... " ~,"~
, ~ ' " ' ~
20 40 60 PERCENT NBT POSITIVE CELLS
80
Fig. l. Nomogram derived from discriminant analysis of the data. Percentage of NBTpositive cells is plotted on the abscissa and the absolute number of NBT-positive cells on the ordinate. For any given patient, the point of intersection of the two values permits visual categorization of the patient into one of the 4 following groups: group A (Control subjects), group B (viral infection, partially treated bacterial infection, and noninfectious febrile illnesses), group C (untreated bacterial infections), or group D (ineffectively treated bacterial infections).
236
Feigin et al.
The Journal o/ Pediatrics February 1971
Table I I I . Results of retrospective classification of 197 individuals Group (No, o/ individuab) A (20) B (130) C (36) D (11)
A 14 29
B 6 7O
C 0 28
D 0 3
1
9
19
7
0
1
4
6
subjects and 29 to 47 per cent in patients with bacterial infections. O u r results are in general agreement with those of Park and associates 5 and others. 7,a Individuals in every group were significantly different from normal with respect to both percentage and absolute numbers of NBT-positive cells. Although an increase was noted in percentage and absolute number of NBT-positive cells in patients with viral infections or noninfectious illness, they were separable from patients with untreated bacterial infections. However, it was not always possible to distinguish patients with partially but effectively treated bacterial infections from the patients with viral illnesses. Failure to separate these groups was most likely the result of the rapidity with which the percentage of NBT-positive cells falls toward normal with effective antibiotic therapy. Patients with bacterial infections who received antibiotic therapy which was inappropriate for their disease developed a greater increase in percentage and absolute number of NBT-positive cells than individuals in any other group. These findings suggest that the number of NBT-positive cells in patients with bacterial infection m a y depend in part upon the duration of the disease process prior to the performance of the N B T test. The increase in percentage of NBT-positive cells noted in the patients sampled is most likely the result of metabolic changes within the white blood cells of the host as a result of the various infectious or inflammatory processes encountered. Wachstein 9 first called attention to the increase in alkaline phosphatase activity of h u m a n neutro-
philic leukocytes during bacterial infections. T h a t rickettsial and viral infections m a y also be associated with leukocyte alkaline phosphatase changes has been documented more recently. I~ Thus, the appearance of increased numbers of NBT-positive cells in association with the various groups of febrile disorders is not unexpected. Moreover, the elevated N B T values noted within 24 hours of onset of clinical symptoms in some patients is not surprising when one considers that leukocyte enzyme changes have been reported to occur in association with infection but prior to the appearance of overt symptoms. 1~ It would appear that the white blood cell response as regards N B T dye reduction is qualitatively similar but quantitatively different in the various patient groups. The white blood cell count is frequently utilized as an aid in the differential diagnosis of infectious diseases. AIthough leukocytosis is frequently associated with bacterial infection, it is also known to accompany such stressful situations as seizures, emotional disturbances, strenuous exercise, burns, or diabetic ketoacidosis? 1 It has previously been shown that leukocyte enzyme changes during episodes of human infection are unrelated to the appearance or disappearance of leukocytosis, l~ Thus, they m a y be a more accurate reflection of infection as opposed to other nonspecific stressful situations. During the course of the present study, the relationship of the percentage of NBTpositive cells to both white blood cell count and body temperature was evaluated. The finding that no correlation existed in most cases serves to re-emphasize the independence of cellular enzyme changes and white biood celi count during h u m a n infection. The exact mechanisms for increased N B T dye reduction have not been ascertained by the present study. Baehner and Nathan 4 noted that decreased N A D H oxidase activity was associated with decreased oxygen consumption and with a diminished ability to reduce N B T dye. Increased leukocyte oxygen consumption and N B T dye reduction has been observed recently in normal
Volume 78 Number 2
newborn infants. 7' 12, 13 Whether increased N B T dye reduction is the result of enhanced N A D H oxidase activity and increased oxygen consumption associated with these febrile disorders or is the result of increased activity of other cellular enzyme systems remains a subject for future investigation. Use of the N B T dye test for patient classification in the present study was of much greater reliability than classification on the basis of simultaneously performed white blood cell counts. In this study, as in that of H u m bert and associates 7 m a n y individuals had relative leukopenia with documented bacterial infections and an unavoidably low absolute number of NBT-positive cells but with a high percentage of positive cells. Conversely, some individuals with leukocytosis and no bacterial infection had an unavoidably high absolute number of NBT-positive neutrophils, but the percentage of N B T positive neutrophils was close to normal. Since both situations m a y obtain, the greatest degree of predictability is achieved by utilizing percentage of NBT-positive cells, as well as the white count-dependent absolute number in deriving the various discriminant functions and the nomogram. Prospective studies to further evaluate the accuracy of the discriminant analysis presented appear to be warranted. T h e simplicity of the N B T dye test utilized in these studies, as well as the results obtained, indicates that this is a useful aid in differentiating patients with febrile disorders. It is apparent, however, that classification of every patient on the basis of the N B T dye test without regard to the patient's history, physical findings, or ancillary laboratory data cannot be accomplished with perfect reliability. The authors wish to thank Mr. Mark Throdahl, Mrs. Hilary Thirkill, and Drs. Thomas MiMe and Richard Wyatt for their assistance. The help of Mrs. Gwendolyn Bailey in the
N B T test in [ebrile disorders
237
preparation of the manuscript is sincerely appreciated, We express our sincere appreciation to Dr. Philip R. Dodge for his critical appraisal of the manuscript and helpful suggestions. REFERENCES
1. Holmes, B., Quie, P. G., Windhorst, D. B., and Good, R. A.: Fatal granulomatous disease of childhood: Inborn abnormality of phagocytic function, Lancet 1: 1225, 1966. 2. Quie, P. G., White, J. G., Holmes, B., and Good, R. A.: In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood, J. Clin. Invest. 46: 668, 1967. 3. Holmes, B., Page, A. R., and Good, R. A.: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function, J. Clin. Invest. 46: 1422, 1967. 4. Baehner, R. I., and Nathan, D. G.: Leukocyte oxidase: Defective activity in chronic granulomatous disease, Science 155: 835, 1967. 5. Park, B. H., Fikrig, S. M., and Smithwick, E. M.: Infection and nitroblue-tetrazolium reduction by neutrophils, Lancet 2: 532, 1968. 6. Anderson, T. W.: An introduction to multivariate statistical analysis, New York, 1957, John Wiley & Sons, Inc., pp. 126-153. 7. Humbert, J. R., Kurtz, M., and Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45: 125, 1970. 8. Matula, G., and Paterson, P. Y.: Spontaneous in vitro nitroblue tetrazolium reduction: A discriminatory test for bacterial infection in adults, J. Clin. Invest. 49: 62a, 1970. (Abst.) 9. Wachstein, M. : Alkaline phosphatase activity in normal and abnormal human blood and bone marrow cells, J. Lab. Clin. Med. 31: 1, 1946. 10. Beisel, W. R.: Neutrophile alkaline phosphatase changes in tularemia, Sandfly fever, Q fever and non-infectious fevers, Blood 29: 257, 1967. 11. Wintrobe, M. M.: Diagnostic significance of changes in leukocytes, Bull. N. Y. Acad. Med. 15: 223, t939. 12. Park, B. H., Holmes, B., and Good, R. A.: Metabolic activities in leukoeytes of newborn infants, J. PEDIAT.76: 237, 1970. 13. Coechi, P., Mori, S., and Becattinl, A.: N.B.T. tests in premature infants, Lancet 2: 1426, 1969.
February, 1971 T h e Journal o/ P E D I A T R I C S
Nitroblue tetrazolium dye test as an aid in the differential diagnosis of febrile disorders Nitroblue tetrazolium (NBT) dye tests were performed upon blood obtained at time of admission or during the course of hospitalization from 247 febrile patients and 20 afebrile individuals. Detailed clinical and laboratory diagnostic evaluation permitted subsequent placement of 181 of the febrile patients into 8 specific groups. Knowledge of only percentage and absolute number of NBT-positive cells permitted reclassification into 4 groups as [oUows: (l) normal subjects, (2) individuals with viral infection, noninfectious fevers, or partially treated bacterial infection, (3) untreated bacterial infection, and (4) bacterial infection unresponsive to therapy provided. There was generally no correlation between percentage of NBT-positive cells and either white blood cell counts or body temperature recorded at the time blood [or the NBT test was obtained. Discriminant analysis was used to prepare a nomogram which permits classification of patients prospectively into one of the 4 groups, if the percentage and absolute number of NBT-positive cells are known.
Ralph D. Feigin, M.D., ~ Penelope G. Shackelford, M.D., Sung C. Choi, Ph.D., Kathryn K. Flake, M.D., Frank A. Franklin, Jr., M.D., and Carl S. Eisenberg, M.D. ST.
LOUIS~
i'VfO.
N E U T R O P H I L S from patients with chronic granulomatous disease of childhood have a diminished bactericidal capacity? Studies of the function and metabolism of these leukocytes have shown that although
From the Department of Pediatrics, and the Division of Biostatistics, Washington University School of Medicine, and the Division of Infectious Diseases, St. Louis Children's Hospital. Reprint address: Dr. Feigln, St. Louls Children's Hospital. 500 S. Kingshi~hway, St. Louis, Mo. 63110. ~'Reeipient of United States Public Health Service Research Career Development Award No. 1KO4A146206 from the National Institute o] Allergy and Infectious Diseases. Supported in part by a Public Health Service Training Grant No. AH00068-05.
Vol. 78, No. 2, pp. 230-237
they ingest bacteria normally,2 intracellular killing of organisms is delayed,a During the course of studies of the respiratory response of Ieukocytes to phagocytosis in this disease, it was noted that the cells failed to reduce nitroblue tetrazolium (NBT) dye.4 Baehner /
See Editor's column, p. 376. and Nathan * attributed this defect to a deficiency of nicotinamide adenine dinucleotide reduced (NADH) oxidase and proposed that the NBT test could be used to detect patients with chronic granulomatous disease. A large number of normal peripheral neu-
Volume 78 Number 2
trophils will reduce NBT dye during phagocytosis in vitro, whereas only small numbers of neutrophils reduce this dye spontaneously. During the course of a bacterial infection in a person with normal neutrophil function, the infecting organism undergoes phagocytosis by neutrophils in vivo. Park and associates s proposed that bacterial infection could induce sufficient metabolic changes in these neutrophils to result in spontaneous in vitro reduction of NBT dye by an increased proportion of neutrophils when compared to healthy control subjects or to patients with nonbacterial disease. They tested this postulate and reported that both the percentage and absolute number of NBT-positive cells were significantly increased in bacterial infection when compared to normal individuals or patients with nonbacterial illnesses. Moreover, there was almost no overlap in the percentage or absolute number of NBTpositive cells in patients with bacterial infection when compared with all other groups. These results suggested that this test might be a simple and useful method for differentiating bacterial infection from nonbacterial disease. Although the results of Park and associates s were of great interest, the precise statistical significance of differences between groups was not presented. In addition, the nurnber of patients with bacterial infections, exclusive of those with meningitis, was small (6),, suggesting that if a larger number of these patients were sampled, greater overlap between some patients with bacterial infection and those in other groups might be observed. The potential effect of previous antibiotic therapy upon the result of the NBT dye test in patients with bacterial infection was not assessed, and the rapidity with which the N B T dye test might return to normal following effective therapeutic intervention was not stated. Several other questions remained unanswered including: (1) Is there a positive, negative, or no correlation between total white blood cell count and percentage of NBT-positive cells? (2) Is the NBT dye test superior to a routine white blood cell count in helping to dis-
N B T test in [ebrile disorders
23 1
tinguish between bacterial and nonbacterial diseases? (3) Does the NBT test have a positive, negative, or no correlation with body temperature at the time the test is performed? (4) With what degree of predictability can one categorize the individual febrile patient if the percentage and absolute number of NBT-positive cells is known ? The usefulness of the NBT dye test as a routine diagnostic aid in the evaluation of patients with infection is dependent upon obtaining answers to these and other similar questions. The present study was undertaken in an attempt to provide this information, as well as to extend previous observations to a larger number of individuals. MATERIAL
AND METHODS
Patient selection. Heparinized blood (1 to 2 ml.) was obtained from 247 patients at the time of admission or during the course of their hospitalization for a febrile illness. For the purposes of this study, patients were considered febrile if a temperature of 101 ~ F. or greater, rectally, was sustained for at least 12 hours or a similar temperature elevation was recorded on at least 3 consecutive days. Heparinized blood (1 to 2 ml.) was also obtained from 20 afebrile control patients (group 1) who had been electively admitted for minor surgical procedures. All patients were between 3 months and 15 years of age. Patients admitted with a febrile illness were the recipients of a detailed diagnostic evaluation which permitted the subsequent placement of 181 of these individuals into specific groups. Patients categorized as having bacterial meningitis (group 2) included only those individuals in whom the diagnosis was established by positive culture of pathogenic bacteria from the cerebrospinal fluid. Individuals categorized as "other bacterial infections" (group 3) included only patients with a well-recognized clinical syndrome, i.e., osteomyelitis, septic arthritis, pharyngitis, pneumonia, impetigo, etc., and from whom a pathogenic bacterium was isolated from the site of infection or the blood. Several patients with bacterial meningitis and other
232
Feigln et aI.
untreated bacterial infections were followed with serial NBT dye tests to determine the rapidity with which the number of NBTpositive ceils returned to normal. Patients classified as having a pretreated bacterial infection (group 4) included individuals with similar types of culturally identified meningeal or nonmeningeal infections but who had received antibiotic therapy prior to the performance of the NBT dye test. In addition, the antibiotic therapy was appropriate for the disease as determined by dose, route of administration, and sensitivity testing for the organism isolated. Patients considered to have noninfectious fevers (group 5) were those with a diagnosis of juvenile rheumatoid arthritis, systemic lupus erythematosus, granulomatous colitis, Letterer-Siwe disease, or chemical pneumonitis determined by appropriate clinical and laboratory criteria. In addition, evidence for concurrent bacterial or viral infection was sought but not found. Individuals categorized as having viral meningitis (group 6) included only patients with clinical signs of meningeal irritation, no previous antibiotic therapy, cerebrospinal fluid changes consistent with the diagnosis, and complete resolution of the disease process without antibiotic therapy. Patients classified as having other viral infections (group 7) included only those individuals with a readily recognizable viral syndrome, i.e., mumps, rubeola, rubella, varicella, herpes simplex, etc., or other individuals from whom a specific virus was isolated and deemed to be of clinical significance because of a fourfold or greater rise in antibody titer to that virus. Patients with ineffectively pretreated bacterial infections (group 8) included those with culturally identified bacterial diseases who were receiving an antibiotic which was deemed ineffective by sensitivity testing for the organism isolated. Four febrile patients with fungal diseases were identified, 2 with pulmonary histoplasmosis, 1 with esophageal moniliasis, and 1 with chronic mucocutaneous moniliasis. Recording of data. Special forms were
The Journal o/ Pediatrics February 1971
prepared to facilitate subsequent computer analysis of the data and 43 specific variables recorded for each patient. Information recorded of specific importance with regard to this report included patient's age, clinical diagnosis, cultural or serologic evidence of infection, previous antibiotic therapy including its duration prior to NBT dye test and its efficacy, duration of symptomatology prior to performance of the NBT dye test, body temperature at the time the NBT dye test was performed, percentage and absolute number of NBT-positive cells, and results of white blood cell and differential counts. Laboratory methods. The NBT dye test was performed as reported by Park and associates? Wright-stained smears were examined under the microscope with oil, and 100, 200, or 500 neutrophils were counted. Since evaluation of this method during the first 4 months of this study indicated that separate results were in good agreement, the percentage of NBT-positive cells was subsequently determined on the basis of a 100 cell count. Only those neutrophils with a large black deposit were considered to be NBT positive. At the same time that blood was obtained for the NBT dye test, a total white blood cell count and differential count was performed. The absolute number of nentrophils per cubic millimeter was calculated. The absolute number of NBT-positive neutrophils was determined from the absolute number of neutrophils and the percentage of NBT-positive cells. Performance of this test can be accomplished within 1 to 2 hours of the time that blood is obtained. Statistleal analysis. Statistical analysis was performed by the Division of Biostatistics, Washington University School of Medicine. Possible differences between the 8 groups in terms of percentage of NBT-positive cells as well as absolute number of NBT-positive cells were investigated by the least significant difference test of which the first stage is analysis of variance. Individual group comparisons were investigated as in the second stage of the least significant difference test by the appropriate t test.
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N B T test in febrile disorders
The potential effect of total white blood cell count and temperature on percentage of NBT-positive cells was studied by computing the correlation coefficients between total white blood cell count and percentage of NBT-positive cells, and temperature and NBT-positive cells for each group. Before analysis, total white blood cell count and temperature were transformed by taking logarithms, and the angular transformation was employed on the percentage of NBTpositive cells to satisfy the statistical assumptions. To determine the degree of predictability with which one can categorize the individual patient if both the percentage and absolute number of NBT-positive cells is known, discriminant analysis was employed. ~ RESULTS
The percentage and absolute number of NBT-positive cells, as well as the standard deviation and ranges for each group, are shown in Table I. It is apparent that both percentage and absolute number of NBTpositive cells are increased in all groups above normal control subjects with the ex-
233
ception of patients with fungal diseases (group 9). Individuals with fungal disease were excluded from further statistical consideration because of their small number. Analysis of variance indicated that there were significant differences between the 8 groups in terms of both percentage of NBT-positive cells (p < 0.001) and absolute number of NBT-positive cells (p < 0.001). On the basis of the percentage and absolute number of NBT-positive cells, all patients tested can be placed into one of 4 larger groups. Afebrile control individuals (group A) differ significantly (p < 0.001) from patients in all other groups and have the lowest mean values. Patients with treated bacterial infection, viral meningitis, other viral infections, and noninfectious fevers do not differ significantly from each other (group B), but significant differences (p < 0.001) are noted when these patients are compared to all other groups. Patients with bacterial meningitis or other bacterial infections (group C) are similar to each other but differ significantly (p < 0.001) from all other groups, whereas individuals with in-
Table I. Results of nitroblue tetrazolium dye test in 181 febrile patients and in 20 control individuals Mean % NBTpositive cells +-ISD 8.75 + 5.25
Ran~.e 2-17
Mean absolute No. I NBT-positive cells +- ISD 420.5 + 278.0
Range 69-949
Group No. 1 Control subjects
No. 20
2 Bacterial meningitis
8
37.1 -+ 16.8
18-70
4331.5 -+ 3237.6
167-9392
3 Other bacterial infections
28
41.3 + 14.9
6-71
3992.7 + 2776.9
784-8760
4 Pretreated bacterial infections
56
22.7 + I8.35
1-60
2358.9 -+ 2 6 1 6 . 4
87-5672
5 Noninfectious fevers
17
14.5 + 13.7
4-42
1706
6 Viral meningitis
24
18.8 + 12.3
2-55
1124.5 -+ 7 4 5 . 4
7 Other viral infections
33
20.2 + 15.6
2-60
1095
-+ 1209.1
8 Pretreated bacterial (ineffective)
11
54.9 + 14.4
31-72
4850
+ 4195
1317-16043
4
8.0 -+ 10.0
2-23
529
-+ 748
130-1650
9 Fungal infections
-+ 2 2 0 2
145-8940 173-2970 34-4516
234
Feigin et al.
The Journal o[ Pediatrics February 1971
effectively treated bacterial infection (group D) differ from all other groups (p < 0.001). T h e correlation coefficients between percentage of NBT-positive cells and either total white bloGd cell count or body temperature are generally small and with one exception not significant at the 5 per cent level based on appropriate degrees of freedom. There is a significant correlation (p < 0.01) between total white blood cell count and percentage of NBT-positive cells in patients with treated bacterial infections. I n all other groups the percentage of N B T positive cells is independent of both the total white blood ceil count and body temperature recorded at the time that blood for the N B T test was obtained. T h e period of time necessary for the percentage of NBT-positive cells to increase above normal was impossible to assess. In some individuals with bacterial meningitis sampled within 12 hours of onset of clinical symptoms, the percentage of NBT-positive cells was already over 30. I n others with bacterial infection and well-documented symptoms for 4 to 5 days, the percentage of NBT-positive cells was similar. T h e rapidity with which the percentage of NBT-positive cells can return to normal is perhaps best illustrated by a 15-monthold patient with Hemophilus influenzae meningitis followed with serial evaluation (Table I I ) . It is apparent that effective antibacterial therapy for as short a time as 18 hours can dramatically reduce the percentage of NBT-positive cells toward normal. Independence of the percentage of NBT-positive cells and the total white blood cell count is also noted. T h e patient's cerebrospinal fluid was sterile after the first 36
hours of therapy. T h e marked elevation in white cell count noted at 114 hours was associated with the development of a subdural effusion which proved to be sterile. The development of this effusion did not affect the percentage of NBT-positive cells which continued to fall to within the normal range. To enhance the clinical applicability of the results obtained, discriminant analysis was employed utilizing the assumption that efficiency of prediction would be greater if one utilized both the percentage and absolute n u m b e r of NBT-positive cells. A discriminant function was obtained representing each of the 4 basic groups of individuals. T h e y are: for group A f1 = -66.8 - 0.71 (% NBT) - 0.019 (Abs. NBT) + 58.5 (log10 Abs. NBT) + 0.00023 (% NBT x Abs. NBT), for group B f, z -76.2 - 0.68 (% NBT) - 0.019 (Abs. NBT) + 61.8 (Iog10 Abs. NBT) + 0.00023 (% NBT x Abs. NBT), for group C f3 = -78.8 - 0.58 (% NBT) - 0.018 (Abs. NBT) + 61.4 (log10 Abs. NBT) + 0.00022 (% NBT x Abs. NBT), and for group D f~ = -84.7 - 0.56 (% NBT) - 0.020 (Abs. NBT) + 62.8 (log~0 Abs. NBT) + 0.00026 (% NBT x Abs. NBT). T h e percentage and absolute n u m b e r of NBT-positive cells obtained for any given individual can be substituted appropriately in each equation. T h e probability that an individual is a m e m b e r of each group can be computed from fl, f2, fa, and f4, and he
Table II. Hemophilus influenzae meningitis
Time o[ treatment Pretreatment After therapy 18 hr. 42 hr. 11~4 hr.
I NBT-positive cells Absolute No. NBT1 (%) positive cells 52 7,972 28 14 8
2,848 1,180 1,066
Total white blood cell count 21,600
% neutrophils 71
18,500 14,800 34,200
55 57 39
Volume 78 Number 2
N B T test in /ebrile disorders
patients is shown in Table I I I . For example, if only the percentage and absolute number of NBT-positive cells were known for the 20 afebrile control individuals (group A), 14 could have been classified correctly while 6 would have been misclassified to group B. None would have been placed in group C or D, groups representing untreated or ineffectively treated bacterial infections, respectively. All misclassified afebrile individuals are o n the border line between group A and Group B when the percentage and absolute number of NBT-positive cells are plotted on the nomogram.
is best classified as a member of the group represented by the largest f value. To facilitate the placement of any given patient into a specific group, a nomogram (Fig. 1) has been prepared based upon the 4 equations which were derived from the data. For any given patient, the point of intersection of the percentage of NBT-positive cells and absolute number of NBT-positive cells permits visual categorization of the patient into one of the 4 groups (A, B, C, D). Probability of correct classification increases as the distance of the point from any dividing line increases. Conversely, if the point of intersection lies near a dividing line, the probability of correct classification decreases. If the point lies precisely on a dividing line, there is an equal probability that the patient should be classified in the groups on either side of the line. The efficiency of the classification procedure when applied retrospectively to our
DISCUSSION Park and associates s have previously reported that the nitroblue tetrazolium dye test might be useful in differentiating patients with bacterial infection from individuals with nonbacterial disease. They showed that the mean proportion of NBT-positive cells was 8.5 per cent in healthy control
I~ooo
• 8,000 x~ 6,000
4,000
k
2.oo0 'K
0 ~
0
", :
-~.~
235
.
-~
~
...... " ~,"~
, ~ ' " ' ~
20 40 60 PERCENT NBT POSITIVE CELLS
80
Fig. l. Nomogram derived from discriminant analysis of the data. Percentage of NBTpositive cells is plotted on the abscissa and the absolute number of NBT-positive cells on the ordinate. For any given patient, the point of intersection of the two values permits visual categorization of the patient into one of the 4 following groups: group A (Control subjects), group B (viral infection, partially treated bacterial infection, and noninfectious febrile illnesses), group C (untreated bacterial infections), or group D (ineffectively treated bacterial infections).
236
Feigin et al.
The Journal o/ Pediatrics February 1971
Table I I I . Results of retrospective classification of 197 individuals Group (No, o/ individuab) A (20) B (130) C (36) D (11)
A 14 29
B 6 7O
C 0 28
D 0 3
1
9
19
7
0
1
4
6
subjects and 29 to 47 per cent in patients with bacterial infections. O u r results are in general agreement with those of Park and associates 5 and others. 7,a Individuals in every group were significantly different from normal with respect to both percentage and absolute numbers of NBT-positive cells. Although an increase was noted in percentage and absolute number of NBT-positive cells in patients with viral infections or noninfectious illness, they were separable from patients with untreated bacterial infections. However, it was not always possible to distinguish patients with partially but effectively treated bacterial infections from the patients with viral illnesses. Failure to separate these groups was most likely the result of the rapidity with which the percentage of NBT-positive cells falls toward normal with effective antibiotic therapy. Patients with bacterial infections who received antibiotic therapy which was inappropriate for their disease developed a greater increase in percentage and absolute number of NBT-positive cells than individuals in any other group. These findings suggest that the number of NBT-positive cells in patients with bacterial infection m a y depend in part upon the duration of the disease process prior to the performance of the N B T test. The increase in percentage of NBT-positive cells noted in the patients sampled is most likely the result of metabolic changes within the white blood cells of the host as a result of the various infectious or inflammatory processes encountered. Wachstein 9 first called attention to the increase in alkaline phosphatase activity of h u m a n neutro-
philic leukocytes during bacterial infections. T h a t rickettsial and viral infections m a y also be associated with leukocyte alkaline phosphatase changes has been documented more recently. I~ Thus, the appearance of increased numbers of NBT-positive cells in association with the various groups of febrile disorders is not unexpected. Moreover, the elevated N B T values noted within 24 hours of onset of clinical symptoms in some patients is not surprising when one considers that leukocyte enzyme changes have been reported to occur in association with infection but prior to the appearance of overt symptoms. 1~ It would appear that the white blood cell response as regards N B T dye reduction is qualitatively similar but quantitatively different in the various patient groups. The white blood cell count is frequently utilized as an aid in the differential diagnosis of infectious diseases. AIthough leukocytosis is frequently associated with bacterial infection, it is also known to accompany such stressful situations as seizures, emotional disturbances, strenuous exercise, burns, or diabetic ketoacidosis? 1 It has previously been shown that leukocyte enzyme changes during episodes of human infection are unrelated to the appearance or disappearance of leukocytosis, l~ Thus, they m a y be a more accurate reflection of infection as opposed to other nonspecific stressful situations. During the course of the present study, the relationship of the percentage of NBTpositive cells to both white blood cell count and body temperature was evaluated. The finding that no correlation existed in most cases serves to re-emphasize the independence of cellular enzyme changes and white biood celi count during h u m a n infection. The exact mechanisms for increased N B T dye reduction have not been ascertained by the present study. Baehner and Nathan 4 noted that decreased N A D H oxidase activity was associated with decreased oxygen consumption and with a diminished ability to reduce N B T dye. Increased leukocyte oxygen consumption and N B T dye reduction has been observed recently in normal
Volume 78 Number 2
newborn infants. 7' 12, 13 Whether increased N B T dye reduction is the result of enhanced N A D H oxidase activity and increased oxygen consumption associated with these febrile disorders or is the result of increased activity of other cellular enzyme systems remains a subject for future investigation. Use of the N B T dye test for patient classification in the present study was of much greater reliability than classification on the basis of simultaneously performed white blood cell counts. In this study, as in that of H u m bert and associates 7 m a n y individuals had relative leukopenia with documented bacterial infections and an unavoidably low absolute number of NBT-positive cells but with a high percentage of positive cells. Conversely, some individuals with leukocytosis and no bacterial infection had an unavoidably high absolute number of NBT-positive neutrophils, but the percentage of N B T positive neutrophils was close to normal. Since both situations m a y obtain, the greatest degree of predictability is achieved by utilizing percentage of NBT-positive cells, as well as the white count-dependent absolute number in deriving the various discriminant functions and the nomogram. Prospective studies to further evaluate the accuracy of the discriminant analysis presented appear to be warranted. T h e simplicity of the N B T dye test utilized in these studies, as well as the results obtained, indicates that this is a useful aid in differentiating patients with febrile disorders. It is apparent, however, that classification of every patient on the basis of the N B T dye test without regard to the patient's history, physical findings, or ancillary laboratory data cannot be accomplished with perfect reliability. The authors wish to thank Mr. Mark Throdahl, Mrs. Hilary Thirkill, and Drs. Thomas MiMe and Richard Wyatt for their assistance. The help of Mrs. Gwendolyn Bailey in the
N B T test in [ebrile disorders
237
preparation of the manuscript is sincerely appreciated, We express our sincere appreciation to Dr. Philip R. Dodge for his critical appraisal of the manuscript and helpful suggestions. REFERENCES
1. Holmes, B., Quie, P. G., Windhorst, D. B., and Good, R. A.: Fatal granulomatous disease of childhood: Inborn abnormality of phagocytic function, Lancet 1: 1225, 1966. 2. Quie, P. G., White, J. G., Holmes, B., and Good, R. A.: In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood, J. Clin. Invest. 46: 668, 1967. 3. Holmes, B., Page, A. R., and Good, R. A.: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function, J. Clin. Invest. 46: 1422, 1967. 4. Baehner, R. I., and Nathan, D. G.: Leukocyte oxidase: Defective activity in chronic granulomatous disease, Science 155: 835, 1967. 5. Park, B. H., Fikrig, S. M., and Smithwick, E. M.: Infection and nitroblue-tetrazolium reduction by neutrophils, Lancet 2: 532, 1968. 6. Anderson, T. W.: An introduction to multivariate statistical analysis, New York, 1957, John Wiley & Sons, Inc., pp. 126-153. 7. Humbert, J. R., Kurtz, M., and Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45: 125, 1970. 8. Matula, G., and Paterson, P. Y.: Spontaneous in vitro nitroblue tetrazolium reduction: A discriminatory test for bacterial infection in adults, J. Clin. Invest. 49: 62a, 1970. (Abst.) 9. Wachstein, M. : Alkaline phosphatase activity in normal and abnormal human blood and bone marrow cells, J. Lab. Clin. Med. 31: 1, 1946. 10. Beisel, W. R.: Neutrophile alkaline phosphatase changes in tularemia, Sandfly fever, Q fever and non-infectious fevers, Blood 29: 257, 1967. 11. Wintrobe, M. M.: Diagnostic significance of changes in leukocytes, Bull. N. Y. Acad. Med. 15: 223, t939. 12. Park, B. H., Holmes, B., and Good, R. A.: Metabolic activities in leukoeytes of newborn infants, J. PEDIAT.76: 237, 1970. 13. Coechi, P., Mori, S., and Becattinl, A.: N.B.T. tests in premature infants, Lancet 2: 1426, 1969.