A
M o d i f i e d T e s t
L e u k o c y t e in A c u t e
N i t r o b l u e
Bacterial
T e t r a z o l i u m
Infection
ROGER M. MILLER, M.D., JOEL GARBUS, P H . D . , ANDREW R. SCHWARTZ, M.D., HERBERT L. D U P O N T , M.D., MYRON M. LEVINE, M.D., DAVID F. CLYDE, M.D., AND RICHARD B. HORNICK,
M.D.
From the University of Maryland School of Medicine, Department of Pathology; Division of Infectious D Department of Medicine; Maryland Institute for Emergency Medicine; and the Department of International Medicine, Baltimore, Maryland ABSTRACT
T H E EARLY DETECTION of systemic infec-
tion is often complicated by the similarity of the manifestations of incipient infection to those of noninfectious febrile disorders. This diagnostic problem is especially pertinent to the care of traumatized and postoperative patients. Although many of these Supported in part by the National Institutes of Health Grants GM-15700, 69-2002, and PH-43-66-63 and the Department of the Army Grants DA-49-193MD-2867 and DA-17-67-C-7057. Received October 21, 1975; received revised manuscript December 29, 1975; accepted for publication January 23, 1976. Address reprint requests to Dr. Miller: Department of Pathology, University of Maryland, School of Medicine, 31 S. Greene Street, Baltimore, Maryland 21201.
patients become seriously infected, others have a febrile course without ever developing systemic infection. Therefore, a rapid, simple test to resolve this diagnostic dilemma is highly desirable. The demonstration by Park and associates13 that infections in children are associated with an increased ability of polymorphonuclear (PMN) leukocytes to reduce nitroblue tetrazolium (NBT) dye provided such a test for the population they investigated. Their findings were confirmed by Feigin and co-workers. 5 Several years ago, we began an investigation of the feasibility of using the N B T test as an aid in the diagnosis of disease
905
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Miller, Roger M., Garbus, Joel, Schwartz, Andrew R., DuPont, Herbert L., Levine, Myron M., Clyde, David F., and Hornick, Richard B.: A modified leukocyte nitroblue tetrazolium test in acute bacterial infection. Am J Clin Pathol 66: 905-910, 1976. T h e increased ability of leukocytes to reduce nitroblue tetrazolium (NBT) has been used to detect the presence of systemic bacterial infection. This test has been utilized to evaluate infections and leukocyte dysfunction in children, but has not been extensively applied to traumatized patients or infected volunteers. Moreover, the technic as originally described presented methodologic difficulties. In this study of 889 such patients, a modified N B T test provided excellent differentiation of 63 systemic bacterial infections (NBT score ^ 10%) from non-infectious fevers, local enteric diseases, and certain viral and plasmodial infections (NBT score s 9%). Splenectomy was associated with a transient false-positive score and clinical typhoid fever with a false-negative response. (Key words: Nitroblue tetrazolium (NBT) test; Polymorphonuclear leukocytes; Infection; Trauma.)
906
MILLER ET AL.
Materials and Methods Laboratory Methods A sample of arterial or venous blood is collected in a standard Vacutainer tube (Becton, Dickinson, and Co.) containing sodium edetate (Na 2 EDTA) anticoagulant. T h e blood (0.2 ml) is mixed with an equal volume of 0.2% N B T (Sigma Chemical Co.) in modified Hanks solution 9 at pH 7.4 in a 12 x 75 mm capped plastic tube and incubated in a water bath at 37 C for 30 minutes. Following incubation, the b l o o d - N B T mixture is gently agitated and smears using the slide-coverslip method are carefully made to avoid damaging the leukocytes. T h e smears are air dried, lightly stained with Wright's stain, and examined under the microscope with oil immersion. From each smear, 100 neutrophils are evaluated. Those cells, both mature and juvenile forms, that contain a large, black cytoplasmic deposit of formazan (reduced NBT) are classified "NBT positive" and the percentage of positives or the "score" is recorded. Studies in our laboratory indicated that leukocytes in the blood samples could be maintained for as long as four hours at ambient temperatures in the Vacutainer tubes without alteration of either normal or abnormal N B T counts, thus allowing
transportation of blood samples from other sites to a single central laboratory for testing. Patient Selection The principal study group consisted of severely traumatized patients in the clinical unit of the Maryland Institute for Emergency Medicine of the University of Maryland Hospital. A broad spectrum of infectious processes with respect to both severity and causative organisms was encountered in these patients. Postoperative thoracic surgery patients, mainly post-cardiotomy, treated in the Maryland Institute for Emergency Medicine, were also examined in this study. A second major study group consisted of inmates voluntarily participating in infectious disease studies at the Maryland House of Correction, Jessup, Maryland. The performance of the present study was incidental to larger vaccine and/or drug efficacy studies conducted by the Division of Infectious Diseases. No infection was induced for the purpose of the present investigation. T h e volunteers were employed in the evaluation of vaccine and/or drug efficacy in induced typhoid fever, shigellosis, influenza, rhinoviral infection, malaria, enteropathogenic Escherichia coli diarrhea, and cholera. T h e specific details of many of these investigations involving volunteers have been p u b lished. 2_4,7,8,15 Normal control values were obtained by testing leukocyte N B T activity in the volunteers prior to challenge with the infectious agents. Results The ranges of values of the leukocyte N B T responses for the normal control subjects as well as for all patient categories that produced results in our normal range are presented in Table 1. T h e N B T responses of leukocytes from 261 normal individuals, i.e., inmate volunteers prior to challenge, never exceeded an N B T count
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in severely traumatized adult patients, infected volunteers, and a variety of patients with milder infections or febrile noninfectious states. However, we found that the test as described by Park 13 presented methodologic difficulties, and we accordingly modified it. T h e modified technic described in this paper eliminated these problems and yet provided an effective distinction between acute systemic bacterial infections on the one hand and non-infectious febrile conditions, viral and malarial infections, and limited enteric bacterial infection on the other.
A.J.C.P. —Vol. 66
November 1976
NBT TEST IN INFECTION
907
Table 1. Conditions Associated with Normal Nitroblue Tetrazolium Test Results
Group
Number of Determinations
Range for Percentage of Positive NBT Cells
261 297
274 1325
114
331
3-9
109 8 8 36 21 20
515 26 16 147 138 36
2-10* 2-9 2-7 3-9 3-8 4-9
9 6
30 17
1-9 2-10*
3-8 5-9
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Control Trauma Thoracic and cardiovascular surgery Shigellosis and/or Shigella vaccine E. coli diarrhea Cholera Typhoid fever Rhinovirus Influenza Malaria Vivax falciparum!
Number of Cases
* Alllinugh l\vn of lilt- injured palients and lluee of the men pai'licipating in die shigellosis invesliganons each had an NUT count of ItKJ in a single insianie, all had NBT scores of 97t or less upon letesdne,. of 9%. Of the 2,855 determinations in blood specimens from 889 individuals, only those of two of the injured patients and three of the men participating in the shigellosis investigations each had an NBT count of 10% in a single instance, with N B T scores of 9% or less upon retesting. Consequently, NBT scores of 9% or less were considered normal. The initial abnormal leukocyte NBT responses in blood samples from infected patients and the associated conditions are shown in Table 2. The initial positive value was defined as that abnormal value that occurred immediately preceding or concomitant with the onset of infection, and followed at least one normal value associated with no infection. This permitted a clear discrimination between NBT-positive-infected patients and NBTnegative-uninfected patients. Only the initial abnormal value is reported because the subsequent values introduced bias into the data. These subsequent values tended to increase until the initiation of proper, specific antimicrobial therapy, which resulted in a trend of decreasing values. An initial leukocyte NBT response of 10% or more was usually associated with a systemic infection (Table 2).
Therefore, scores of 10% or more were categorized as abnormal. In the absence of infection, the samples from 297 traumatized patients and the 114 postoperative thoracic surgery patients had normal NBT scores (Table 1). Normal NBT values were also seen in those enteric diarrheal diseases associated with (1) enteropathogenic E. coli and Vibrio cholerae in which the disease is due primarily to the local effect of a toxin, and (2) mild shigellosis and/or exposure to attenuated oral shigella vaccine, where the diarrheal syndrome is produced by local, limited infection confined to the intestinal mucosa. These diarrheal agents characteristically do not manifest systemic invasion, although increases of humoral antibody titers to the specific strain were found in many cases. Although viral diseases have been reported to be associated with increased N B T counts, 2 we did not obtain elevated counts in 21 cases of rhinovirus infection or in 20 cases of influenza, despite the presence of marked systemic clinical responses in most instances. It has also been reported that Plasmodium vivax malaria will produce a positive NBT test.10 However, utilizing the present technic, specimens from nine patients with Plasmodium
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A.J.C.P. —Vol. 66
dialysis. Slight but significant elevations of N B T scores were associated with either well-localized or actively draining infections such as localized abscesses, open softPercentage of tissue infections, or urinary tract infections Positive Number NBT Cells* associated with indwelling catheters (Table of 2). Indeed, either proper surgical drainage Group Cases Mean Range of an infected area or the institution of 20.0 10-50 proper antibiotic therapy resulted in a Bacteremia 33 10.9 10-13 marked, prompt decrease in the daily NBT 7 Bronchopneumonitis Sod-tissue infection/ count. Conversely, the persistence of an abscess 13.4 10-19 elevated NBT count suggested inadePeritonitis 33.0 27-37 quate therapy. A slight elevation of the Primary 11.3 10-15 leukocyte N B T score was found for several Secondary to dialysis 10.8 10-12 Urinary tract infection 12.0 11-13 days following splenectomy. No infection Post-splenectomy was documented and antibiotics were not * Initial positive value immediately preceding or concomitant with needed during this time. This situation rethe onset of infection, following at least one normal value associated sulted in the only instances of a "falsewith no infection. positive" response in this series. Table 2. Conditions Associated with Abnormal Nitroblue Tetrazolium Test Results
Discussion T h e present method of performing the leukocyte N B T reduction test eliminates some of the technical problems we encountered with previously described technics. These problems include: (a) drying of the preparation during incubation despite the use of wet gauze to provide humidity, (b) a nonspecific interaction between N B T and heparin that interfered with the performance of the test, (c) the complexity of the procedure, which prevented the efficient processing of large numbers of samples, and (d) difficulty in preparing satisfactory smears for microscopic evaluation. This last technical problem is particularly critical because a valid evaluation of the leukocytes is difficult to obtain when the smears are distorted by clumping of the neutrophils. Thin, easily read blood films that do not contain clustered leukocytes are readily obtained using the present method, which does not require the extensive preparation of slides and chambers previously described. 10 Nitroblue tetrazolium dye has been used to assess leukocytic function under a variety of conditions. During phagocytosis in vitro,
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vivax and six patients with Plasmodium falciparum malaria all had counts in the normal range during the acute infectious phase. No value greater than 9% was seen in blood from 36 volunteers who had clinical typhoid fever, despite documented bacteremia in nine of the 21 men considered ill enough to require more intensive therapy. When feasible, daily leukocyte NBT reduction determinations were performed in all cases of traumatized and postoperative thoracic surgery patients in the Maryland Institute for Emergency Medicine. Although the vast majority of these results were normal (Table 1), positive tests were obtained with blood from patients with systemic infections (Table 2). T h e greatest number of positive tests was associated with bacteremias, which were entirely of gram-negative rod etiology. Peritonitis resulted in markedly elevated N B T counts, which tended to fall rapidly toward normal when drainage by dialysis was started for concomitant renal failure. This reduced count seen in primary peritonitis with subsequent dialysis was comparable in magnitude to that seen in peritonitis occurring as a consequence of peritoneal
November 1976
NBT TEST IN INFECTION
The reduction of nitroblue tetrazolium by PMN leukocytes is a reflection of the activity of a cyanide-insensitive, cytoplasmic, pyridine nucleotide oxidase, 1 an enzyme intimately related to the increased oxygen consumption, increased pentose shunt activity, and increased hydrogen peroxide formation that are associated with phagocytosis, degranulation, and intracellular destruction of bacterial organisms. T h e normal leukocyte N B T scores in blood from volunteers with clinical typhoid fever were unexpected, especially in those cases with documented bacteremia, in which abnormal, elevated N B T responses would be anticipated. This anomalous finding prompted further investigations on our part, and the association of virulent Salmonella typhi with a lack of enhanced stimulation of leukocyte postphagocytic hexose monophosphate shunt activity was demonstrated. 11 This was in contrast to an enhanced rate of oxygen consumption normally observed during phagocytosis of relatively avirulent strains of Salmonella typhi or Staphylococcus aureus. These data suggest that the ability of a bacterial strain to alter the post-phagocytic rate of oxygen consumption of polymorphonuclear leukocytes may be related to its virulence in vivo. T h e roles of NBT-positive cells in the
normal and in the infected person are not well defined. Of great interest is the moderate increase in the N B T count following splenectomy. The specific stimulus that produces cells that may be classified as NBT-positive is not known, although it is believed to be related to pyridine nucleotide oxidase activity associated with the hexose monophosphate shunt. The use of EDTA as an anticoagulant prevents the phagocytosis of extracellular particles of reduced NBT, and suggests that NBT penetrates the leukocyte and is reduced and precipitated intracellularly. T h e increased ability of a dye to penetrate a living cell suggests that such a cell is somehow altered. Since the spleen removes imperfect, dying, and injured cells from circulation as part of its function, the increase in the NBT count after splenectomy suggests that the NBT-positive cells may be imperfect or damaged. The normal range of such cells would be maintained by the action of the spleen in removing them from the circulation. However, rather than representing a manifestation of injury to host cells by the infection, it may reflect enhanced phagocytic or metabolic activity as a par f of the host resistance pattern, and the cells may become marked as altered in the process of serving this function. Injured patients are highly susceptible to systemic infection. 12,14 This enhanced morbidity is associated with a prolonged clinical course and often with death. The ability to distinguish a fever of non-infectious etiology from one caused by severe systemic infection is of obvious importance if the life of a severely injured person is to be saved. The indiscriminate use of antibiotics to "treat" a fever presumed to be of infectious origin may be detrimental to the patient due to the elimination of the normal host bacterial flora that may protect a patient from pathogenic invaders. In addition, excessive use of antibiotics increases the risk of drug toxicity. Postoperative thoracic surgery patients often
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an increased number of normal blood leukocytes reduce NBT dye compared with cells under resting conditions. 6 PMN leukocytes obtained from children affected with chronic granulomatous diseases phagocytize bacteria in vitro but manifest a decreased capacity to kill the ingested organisms. 6 They also show a diminished rate of NBT dye reduction during in-vitro phagocytosis of latex particles. On the other hand, an increased percentage of NBT-positive leukocytes has been observed in the blood of normal children who have acute bacterial infections. 5 ' 13 These data suggest a relationship between the capacity of PMN leukocytes to reduce NBT dye and their bactericidal efficacy.
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66
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strain resistant to quinine. JAMA 213:2041have fevers that are attributed to atelectasis. 2045, 1970 T h e normal N B T tests observed in blood 3. DuPont HL, Formal SB, Hornick RB, et al: Pathogenesis of Escherichia coli diarrhea. N of such patients in this study indicate that Engl J Med 285:1-9, 1971 their fever is not the result of systemic 4. DuPont HL, Hornick RB, Snyder MJ, et al: bacterial infection. T h e marginally abImmunity in shigellosis. II. Effect of oral live vaccine and re-challenge on subsequent disnormal values in blood from those patients ease. J Infect Dis 125:12-16, 1972 with demonstrable pneumonitis in the 5. Feigin RD, Shackelford PG, Choi SC, et al: Nitroblue tetrazolium dye test as an aid in the present series probably reflect the fact that differential diagnosis of febrile disorders. J these patients had "draining" bronchoPediatr 78:230-237, 1971 pneumonia. 6. Holmes B, Page AR, Good RA: Studies of metabolic activity of leukocytes from patients with Our series demonstrates that trauma itgenetic abnormality of phagocytic function. self, whether accidental or elective, as in J Clin Invest 46:1422-1432, 1967 those patients who underwent cardiac or 7. Hornick RB, Greisman SE, Woodward TE, et al: Typhoid fever: Pathogenesis and immunopulmonary surgery, does not influence the logic control. N Engl J Med 283:686-691, 1970 results of the N B T test in spite of the RB, Music SI, Wenzel R, et al: The fact that leukocytosis and/or fever may 8. Hornick Broad Street pump revisited: Response of follow the injury. T h e changes in the daily volunteers to ingested cholera vibrios. Bull NY Acad Med 47:1181-1191, 1971 counts of NBT-positive cells in patients 9. Martin SP, Green R: Methods for the study of who were, or subsequently became, inhuman leukocytes. Methods Med Res 7:137, 1958 fected were an invaluable aid in diagnosis G, Paterson PY: Spontaneous in vitro of the infection and in evaluating efficacy 10. Matula nitroblue tetrazolium reduction by neutrophils of therapy. Therefore, we feel that daily of adult patients with bacterial infection. N EnglJ Med 285:311-316, 1971 evaluations of the N B T test should be in11. Miller RM, Garbus J, Hornick RB: Lack of encluded in the routine diagnostic regimen of hanced oxygen consumption by polymorphonuclear leukocytes on phagocytosis of virulent these patients. Salmonella typhi. Science 175:1010, 1972 All protocols involving volunteers were approved 12. Miller RM, Polakavetz SH, Hornick RB, et al: Analysis of infections acquired by the severely by the University of Maryland Committee on Human injured patient. Surg Gynecol Obstet 137: Experimentation. The ethical guidelines followed in 7-10, 1973 these volunteer studies conformed to the Declaration of Helsinki as approved by the World Medical 13. Park BH, Fikrig SM, Smithwick EM: Infection and nitroblue tetrazolium reduction by Association in 1964. neutrophils. Lancet 2:532-534, 1968 14. Schimpff SC, Miller RM, Polakavetz S, et al: References Infection in the severely traumatized patient. 1. Baehner RI, Nathan DC: Leukocyte oxidase: Ann Surg 179:352-357, 1974 Defective activity in chronic granulomatous 15. Togo Y, Schwartz AR, Hornick RB: Failure of a disease. Science 155:835-836, 1967 3-substituted triazinoindole in the prevention 2. Clyde DF, Miller RM, DuPont HL, et al: of experimental human rhinovirus infection. Treatment of falciparum malaria caused by Chemotherapy 18:17-26, 1973
M o d i f i e d T e s t
L e u k o c y t e in A c u t e
N i t r o b l u e
Bacterial
T e t r a z o l i u m
Infection
ROGER M. MILLER, M.D., JOEL GARBUS, P H . D . , ANDREW R. SCHWARTZ, M.D., HERBERT L. D U P O N T , M.D., MYRON M. LEVINE, M.D., DAVID F. CLYDE, M.D., AND RICHARD B. HORNICK,
M.D.
From the University of Maryland School of Medicine, Department of Pathology; Division of Infectious D Department of Medicine; Maryland Institute for Emergency Medicine; and the Department of International Medicine, Baltimore, Maryland ABSTRACT
T H E EARLY DETECTION of systemic infec-
tion is often complicated by the similarity of the manifestations of incipient infection to those of noninfectious febrile disorders. This diagnostic problem is especially pertinent to the care of traumatized and postoperative patients. Although many of these Supported in part by the National Institutes of Health Grants GM-15700, 69-2002, and PH-43-66-63 and the Department of the Army Grants DA-49-193MD-2867 and DA-17-67-C-7057. Received October 21, 1975; received revised manuscript December 29, 1975; accepted for publication January 23, 1976. Address reprint requests to Dr. Miller: Department of Pathology, University of Maryland, School of Medicine, 31 S. Greene Street, Baltimore, Maryland 21201.
patients become seriously infected, others have a febrile course without ever developing systemic infection. Therefore, a rapid, simple test to resolve this diagnostic dilemma is highly desirable. The demonstration by Park and associates13 that infections in children are associated with an increased ability of polymorphonuclear (PMN) leukocytes to reduce nitroblue tetrazolium (NBT) dye provided such a test for the population they investigated. Their findings were confirmed by Feigin and co-workers. 5 Several years ago, we began an investigation of the feasibility of using the N B T test as an aid in the diagnosis of disease
905
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Miller, Roger M., Garbus, Joel, Schwartz, Andrew R., DuPont, Herbert L., Levine, Myron M., Clyde, David F., and Hornick, Richard B.: A modified leukocyte nitroblue tetrazolium test in acute bacterial infection. Am J Clin Pathol 66: 905-910, 1976. T h e increased ability of leukocytes to reduce nitroblue tetrazolium (NBT) has been used to detect the presence of systemic bacterial infection. This test has been utilized to evaluate infections and leukocyte dysfunction in children, but has not been extensively applied to traumatized patients or infected volunteers. Moreover, the technic as originally described presented methodologic difficulties. In this study of 889 such patients, a modified N B T test provided excellent differentiation of 63 systemic bacterial infections (NBT score ^ 10%) from non-infectious fevers, local enteric diseases, and certain viral and plasmodial infections (NBT score s 9%). Splenectomy was associated with a transient false-positive score and clinical typhoid fever with a false-negative response. (Key words: Nitroblue tetrazolium (NBT) test; Polymorphonuclear leukocytes; Infection; Trauma.)
906
MILLER ET AL.
Materials and Methods Laboratory Methods A sample of arterial or venous blood is collected in a standard Vacutainer tube (Becton, Dickinson, and Co.) containing sodium edetate (Na 2 EDTA) anticoagulant. T h e blood (0.2 ml) is mixed with an equal volume of 0.2% N B T (Sigma Chemical Co.) in modified Hanks solution 9 at pH 7.4 in a 12 x 75 mm capped plastic tube and incubated in a water bath at 37 C for 30 minutes. Following incubation, the b l o o d - N B T mixture is gently agitated and smears using the slide-coverslip method are carefully made to avoid damaging the leukocytes. T h e smears are air dried, lightly stained with Wright's stain, and examined under the microscope with oil immersion. From each smear, 100 neutrophils are evaluated. Those cells, both mature and juvenile forms, that contain a large, black cytoplasmic deposit of formazan (reduced NBT) are classified "NBT positive" and the percentage of positives or the "score" is recorded. Studies in our laboratory indicated that leukocytes in the blood samples could be maintained for as long as four hours at ambient temperatures in the Vacutainer tubes without alteration of either normal or abnormal N B T counts, thus allowing
transportation of blood samples from other sites to a single central laboratory for testing. Patient Selection The principal study group consisted of severely traumatized patients in the clinical unit of the Maryland Institute for Emergency Medicine of the University of Maryland Hospital. A broad spectrum of infectious processes with respect to both severity and causative organisms was encountered in these patients. Postoperative thoracic surgery patients, mainly post-cardiotomy, treated in the Maryland Institute for Emergency Medicine, were also examined in this study. A second major study group consisted of inmates voluntarily participating in infectious disease studies at the Maryland House of Correction, Jessup, Maryland. The performance of the present study was incidental to larger vaccine and/or drug efficacy studies conducted by the Division of Infectious Diseases. No infection was induced for the purpose of the present investigation. T h e volunteers were employed in the evaluation of vaccine and/or drug efficacy in induced typhoid fever, shigellosis, influenza, rhinoviral infection, malaria, enteropathogenic Escherichia coli diarrhea, and cholera. T h e specific details of many of these investigations involving volunteers have been p u b lished. 2_4,7,8,15 Normal control values were obtained by testing leukocyte N B T activity in the volunteers prior to challenge with the infectious agents. Results The ranges of values of the leukocyte N B T responses for the normal control subjects as well as for all patient categories that produced results in our normal range are presented in Table 1. T h e N B T responses of leukocytes from 261 normal individuals, i.e., inmate volunteers prior to challenge, never exceeded an N B T count
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
in severely traumatized adult patients, infected volunteers, and a variety of patients with milder infections or febrile noninfectious states. However, we found that the test as described by Park 13 presented methodologic difficulties, and we accordingly modified it. T h e modified technic described in this paper eliminated these problems and yet provided an effective distinction between acute systemic bacterial infections on the one hand and non-infectious febrile conditions, viral and malarial infections, and limited enteric bacterial infection on the other.
A.J.C.P. —Vol. 66
November 1976
NBT TEST IN INFECTION
907
Table 1. Conditions Associated with Normal Nitroblue Tetrazolium Test Results
Group
Number of Determinations
Range for Percentage of Positive NBT Cells
261 297
274 1325
114
331
3-9
109 8 8 36 21 20
515 26 16 147 138 36
2-10* 2-9 2-7 3-9 3-8 4-9
9 6
30 17
1-9 2-10*
3-8 5-9
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Control Trauma Thoracic and cardiovascular surgery Shigellosis and/or Shigella vaccine E. coli diarrhea Cholera Typhoid fever Rhinovirus Influenza Malaria Vivax falciparum!
Number of Cases
* Alllinugh l\vn of lilt- injured palients and lluee of the men pai'licipating in die shigellosis invesliganons each had an NUT count of ItKJ in a single insianie, all had NBT scores of 97t or less upon letesdne,. of 9%. Of the 2,855 determinations in blood specimens from 889 individuals, only those of two of the injured patients and three of the men participating in the shigellosis investigations each had an NBT count of 10% in a single instance, with N B T scores of 9% or less upon retesting. Consequently, NBT scores of 9% or less were considered normal. The initial abnormal leukocyte NBT responses in blood samples from infected patients and the associated conditions are shown in Table 2. The initial positive value was defined as that abnormal value that occurred immediately preceding or concomitant with the onset of infection, and followed at least one normal value associated with no infection. This permitted a clear discrimination between NBT-positive-infected patients and NBTnegative-uninfected patients. Only the initial abnormal value is reported because the subsequent values introduced bias into the data. These subsequent values tended to increase until the initiation of proper, specific antimicrobial therapy, which resulted in a trend of decreasing values. An initial leukocyte NBT response of 10% or more was usually associated with a systemic infection (Table 2).
Therefore, scores of 10% or more were categorized as abnormal. In the absence of infection, the samples from 297 traumatized patients and the 114 postoperative thoracic surgery patients had normal NBT scores (Table 1). Normal NBT values were also seen in those enteric diarrheal diseases associated with (1) enteropathogenic E. coli and Vibrio cholerae in which the disease is due primarily to the local effect of a toxin, and (2) mild shigellosis and/or exposure to attenuated oral shigella vaccine, where the diarrheal syndrome is produced by local, limited infection confined to the intestinal mucosa. These diarrheal agents characteristically do not manifest systemic invasion, although increases of humoral antibody titers to the specific strain were found in many cases. Although viral diseases have been reported to be associated with increased N B T counts, 2 we did not obtain elevated counts in 21 cases of rhinovirus infection or in 20 cases of influenza, despite the presence of marked systemic clinical responses in most instances. It has also been reported that Plasmodium vivax malaria will produce a positive NBT test.10 However, utilizing the present technic, specimens from nine patients with Plasmodium
908
MILLER ETAL.
A.J.C.P. —Vol. 66
dialysis. Slight but significant elevations of N B T scores were associated with either well-localized or actively draining infections such as localized abscesses, open softPercentage of tissue infections, or urinary tract infections Positive Number NBT Cells* associated with indwelling catheters (Table of 2). Indeed, either proper surgical drainage Group Cases Mean Range of an infected area or the institution of 20.0 10-50 proper antibiotic therapy resulted in a Bacteremia 33 10.9 10-13 marked, prompt decrease in the daily NBT 7 Bronchopneumonitis Sod-tissue infection/ count. Conversely, the persistence of an abscess 13.4 10-19 elevated NBT count suggested inadePeritonitis 33.0 27-37 quate therapy. A slight elevation of the Primary 11.3 10-15 leukocyte N B T score was found for several Secondary to dialysis 10.8 10-12 Urinary tract infection 12.0 11-13 days following splenectomy. No infection Post-splenectomy was documented and antibiotics were not * Initial positive value immediately preceding or concomitant with needed during this time. This situation rethe onset of infection, following at least one normal value associated sulted in the only instances of a "falsewith no infection. positive" response in this series. Table 2. Conditions Associated with Abnormal Nitroblue Tetrazolium Test Results
Discussion T h e present method of performing the leukocyte N B T reduction test eliminates some of the technical problems we encountered with previously described technics. These problems include: (a) drying of the preparation during incubation despite the use of wet gauze to provide humidity, (b) a nonspecific interaction between N B T and heparin that interfered with the performance of the test, (c) the complexity of the procedure, which prevented the efficient processing of large numbers of samples, and (d) difficulty in preparing satisfactory smears for microscopic evaluation. This last technical problem is particularly critical because a valid evaluation of the leukocytes is difficult to obtain when the smears are distorted by clumping of the neutrophils. Thin, easily read blood films that do not contain clustered leukocytes are readily obtained using the present method, which does not require the extensive preparation of slides and chambers previously described. 10 Nitroblue tetrazolium dye has been used to assess leukocytic function under a variety of conditions. During phagocytosis in vitro,
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
vivax and six patients with Plasmodium falciparum malaria all had counts in the normal range during the acute infectious phase. No value greater than 9% was seen in blood from 36 volunteers who had clinical typhoid fever, despite documented bacteremia in nine of the 21 men considered ill enough to require more intensive therapy. When feasible, daily leukocyte NBT reduction determinations were performed in all cases of traumatized and postoperative thoracic surgery patients in the Maryland Institute for Emergency Medicine. Although the vast majority of these results were normal (Table 1), positive tests were obtained with blood from patients with systemic infections (Table 2). T h e greatest number of positive tests was associated with bacteremias, which were entirely of gram-negative rod etiology. Peritonitis resulted in markedly elevated N B T counts, which tended to fall rapidly toward normal when drainage by dialysis was started for concomitant renal failure. This reduced count seen in primary peritonitis with subsequent dialysis was comparable in magnitude to that seen in peritonitis occurring as a consequence of peritoneal
November 1976
NBT TEST IN INFECTION
The reduction of nitroblue tetrazolium by PMN leukocytes is a reflection of the activity of a cyanide-insensitive, cytoplasmic, pyridine nucleotide oxidase, 1 an enzyme intimately related to the increased oxygen consumption, increased pentose shunt activity, and increased hydrogen peroxide formation that are associated with phagocytosis, degranulation, and intracellular destruction of bacterial organisms. T h e normal leukocyte N B T scores in blood from volunteers with clinical typhoid fever were unexpected, especially in those cases with documented bacteremia, in which abnormal, elevated N B T responses would be anticipated. This anomalous finding prompted further investigations on our part, and the association of virulent Salmonella typhi with a lack of enhanced stimulation of leukocyte postphagocytic hexose monophosphate shunt activity was demonstrated. 11 This was in contrast to an enhanced rate of oxygen consumption normally observed during phagocytosis of relatively avirulent strains of Salmonella typhi or Staphylococcus aureus. These data suggest that the ability of a bacterial strain to alter the post-phagocytic rate of oxygen consumption of polymorphonuclear leukocytes may be related to its virulence in vivo. T h e roles of NBT-positive cells in the
normal and in the infected person are not well defined. Of great interest is the moderate increase in the N B T count following splenectomy. The specific stimulus that produces cells that may be classified as NBT-positive is not known, although it is believed to be related to pyridine nucleotide oxidase activity associated with the hexose monophosphate shunt. The use of EDTA as an anticoagulant prevents the phagocytosis of extracellular particles of reduced NBT, and suggests that NBT penetrates the leukocyte and is reduced and precipitated intracellularly. T h e increased ability of a dye to penetrate a living cell suggests that such a cell is somehow altered. Since the spleen removes imperfect, dying, and injured cells from circulation as part of its function, the increase in the NBT count after splenectomy suggests that the NBT-positive cells may be imperfect or damaged. The normal range of such cells would be maintained by the action of the spleen in removing them from the circulation. However, rather than representing a manifestation of injury to host cells by the infection, it may reflect enhanced phagocytic or metabolic activity as a par f of the host resistance pattern, and the cells may become marked as altered in the process of serving this function. Injured patients are highly susceptible to systemic infection. 12,14 This enhanced morbidity is associated with a prolonged clinical course and often with death. The ability to distinguish a fever of non-infectious etiology from one caused by severe systemic infection is of obvious importance if the life of a severely injured person is to be saved. The indiscriminate use of antibiotics to "treat" a fever presumed to be of infectious origin may be detrimental to the patient due to the elimination of the normal host bacterial flora that may protect a patient from pathogenic invaders. In addition, excessive use of antibiotics increases the risk of drug toxicity. Postoperative thoracic surgery patients often
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an increased number of normal blood leukocytes reduce NBT dye compared with cells under resting conditions. 6 PMN leukocytes obtained from children affected with chronic granulomatous diseases phagocytize bacteria in vitro but manifest a decreased capacity to kill the ingested organisms. 6 They also show a diminished rate of NBT dye reduction during in-vitro phagocytosis of latex particles. On the other hand, an increased percentage of NBT-positive leukocytes has been observed in the blood of normal children who have acute bacterial infections. 5 ' 13 These data suggest a relationship between the capacity of PMN leukocytes to reduce NBT dye and their bactericidal efficacy.
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strain resistant to quinine. JAMA 213:2041have fevers that are attributed to atelectasis. 2045, 1970 T h e normal N B T tests observed in blood 3. DuPont HL, Formal SB, Hornick RB, et al: Pathogenesis of Escherichia coli diarrhea. N of such patients in this study indicate that Engl J Med 285:1-9, 1971 their fever is not the result of systemic 4. DuPont HL, Hornick RB, Snyder MJ, et al: bacterial infection. T h e marginally abImmunity in shigellosis. II. Effect of oral live vaccine and re-challenge on subsequent disnormal values in blood from those patients ease. J Infect Dis 125:12-16, 1972 with demonstrable pneumonitis in the 5. Feigin RD, Shackelford PG, Choi SC, et al: Nitroblue tetrazolium dye test as an aid in the present series probably reflect the fact that differential diagnosis of febrile disorders. J these patients had "draining" bronchoPediatr 78:230-237, 1971 pneumonia. 6. Holmes B, Page AR, Good RA: Studies of metabolic activity of leukocytes from patients with Our series demonstrates that trauma itgenetic abnormality of phagocytic function. self, whether accidental or elective, as in J Clin Invest 46:1422-1432, 1967 those patients who underwent cardiac or 7. Hornick RB, Greisman SE, Woodward TE, et al: Typhoid fever: Pathogenesis and immunopulmonary surgery, does not influence the logic control. N Engl J Med 283:686-691, 1970 results of the N B T test in spite of the RB, Music SI, Wenzel R, et al: The fact that leukocytosis and/or fever may 8. Hornick Broad Street pump revisited: Response of follow the injury. T h e changes in the daily volunteers to ingested cholera vibrios. Bull NY Acad Med 47:1181-1191, 1971 counts of NBT-positive cells in patients 9. Martin SP, Green R: Methods for the study of who were, or subsequently became, inhuman leukocytes. Methods Med Res 7:137, 1958 fected were an invaluable aid in diagnosis G, Paterson PY: Spontaneous in vitro of the infection and in evaluating efficacy 10. Matula nitroblue tetrazolium reduction by neutrophils of therapy. Therefore, we feel that daily of adult patients with bacterial infection. N EnglJ Med 285:311-316, 1971 evaluations of the N B T test should be in11. Miller RM, Garbus J, Hornick RB: Lack of encluded in the routine diagnostic regimen of hanced oxygen consumption by polymorphonuclear leukocytes on phagocytosis of virulent these patients. Salmonella typhi. Science 175:1010, 1972 All protocols involving volunteers were approved 12. Miller RM, Polakavetz SH, Hornick RB, et al: Analysis of infections acquired by the severely by the University of Maryland Committee on Human injured patient. Surg Gynecol Obstet 137: Experimentation. The ethical guidelines followed in 7-10, 1973 these volunteer studies conformed to the Declaration of Helsinki as approved by the World Medical 13. Park BH, Fikrig SM, Smithwick EM: Infection and nitroblue tetrazolium reduction by Association in 1964. neutrophils. Lancet 2:532-534, 1968 14. Schimpff SC, Miller RM, Polakavetz S, et al: References Infection in the severely traumatized patient. 1. Baehner RI, Nathan DC: Leukocyte oxidase: Ann Surg 179:352-357, 1974 Defective activity in chronic granulomatous 15. Togo Y, Schwartz AR, Hornick RB: Failure of a disease. Science 155:835-836, 1967 3-substituted triazinoindole in the prevention 2. Clyde DF, Miller RM, DuPont HL, et al: of experimental human rhinovirus infection. Treatment of falciparum malaria caused by Chemotherapy 18:17-26, 1973
