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Performance characteristics of urine dipsticks stored in open containers
Performance Characteristics of Urine Dipsticks Stored in Open Containers E. JOHN GALLAGHER,
Dip and read urinalysis is a laboratory test commonly performed by emergency physicians. Although the manufacturer states that the capped vials contolning the dipsticks must be closed immediately after removal of a strip, this recommendation may not be followed in a busy emergency department. In a simple, two-part, blinded, and controlled trial the authors found that the reagents for determining leukocyte esterase, pH, protein, glucose, ketones, urobilinogen, blllrubin, and blood showed good reproducibility when fresh dipstlcks were compared with dipsticks exposed to room temperature and humidity for 1 to 15 days. In contrast to this, the nitrite portion of the exposed dipsticks showed a rapid and cumulative loss of specificity over time. By the end of a week of exposure, one third of the nitrite tests gave false-positive readings. At the end of a second week, nearly three quarters gave false-positive readings for a specificity of only 28%. It is concluded that the nitrite reagent, in contrast to the other eight reagents on the Chemstrtp-g dipstick (Biodynamics, Indianapolis, IN), rapidly loses accuracy when stored in uncapped vials. (Am J Emerg Med lggO;g:121-123. 0 1990 by W.B. Saunders Company.)
Clinically efficient, cost-effective algorithms using dip and read urinalysis are dependent on dipstick accuracy.‘” Accuracy of reagent strips is contingent on proper storage of these strips according to the manufacturer’s recommendations. A survey of the three physician laboratories in our emergency department (ED) showed that these containers are rarely recapped after removal of a reagent strip. We designed a simple, blinded, controlled trial to determine if exposure of urine dipsticks to room temperature and humidity affects their accuracy over time. METHODS
The Chemstrip-9 (Biodynamics, Indianapolis, IN) is an inert plastic strip made up of a sequence of nine discrete reagent pads for the determination of urinary leukocytes, nitrites, pH, protein, glucose, ketones, urobilinogen, bilirubin, and blood. The strip is immersed in urine for 1 second and read at 1 minute according to a color chart. One hundred sticks are packed in an aluminum vial with a tightly fitting stopper impregnated with a desiccant. In the first part of the study, 20 dipsticks from each of 10 containers left open for 1 to 15 days were used to test 20 urines collected at random in the ED for a total of 200 trials (20 dipstick urinalyses per container x 10 containers = 200 From the *Emergency Medical Services, Ambulatory Care, Bronx Municipal Hospital Center and the tDepartment of Medicine. Albert Einstein College of Medicine, Bronx, NY. Manuscript received April 6, 1969; accepted April 25, 1969. Address reprint requests to Dr Gallagher: Jacobi Hospital, Rm 1 W22, Bronx Municipal Hospital Center, Pelham Parkway S and Eastchester Rd, Bronx, NY 10461. Key Words: Urinalysis, errors in dipstick urinalysis, falsepositive nitrate reagent test. 0 1990 by W.B. Saunders Company. 0735.6757/90/0602-0006$5.00/O
MD,*t EDWARD SCHWARTZ, MD,* ROSLYN S. WEINSTEIN, MED*
trials). Results differing by more than one color shade from the reading of a fresh (control) dipstick were recorded as discordant by an observer who had been blinded to the age of the exposed dipsticks. Based on the findings from the initial portion of the study (Fig 1, solid line), the effect of exposure on the nitrite reagent portion of the dipstick was reexamined, using urine culture results as an external standard. Twenty dipsticks from each of 10 containers left open for 1 to 15 days were used to test urine collected from 20 ambulatory asymptomatic out-patients (200 trials). No patients were taking rifampin, phenazopyridine, or ascorbate,4 which might affect the accuracy of the nitrite test. Only patients with negative nitrite and leukocyte esterase tests were in this part of the study. Urines were subsequently cultured by the hospital laboratory using standard techniques. The observer reading the dipsticks had been blinded to the number of days each container had been exposed to room temperature and humidity. According to the manufacturer’s recommendation, any pink color of the nitrite reagent was recorded as positive. Cultures were considered positive if the urine grew more than 100,000 organisms/mL.5 RESULTS
Eight out of nine reagents showed excellent reproducibility and internal consistency when fresh urine dipsticks were compared with those exposed to room temperature and humidity for 1 to 15 days. The increasing discordance between the fresh and exposed nitrite reagent strips that occurred in 66 out of 68 instances (97%), was associated with a progressive increase in the number of false positives over time (Fig 1, solid line). In the second part of the study, when urine cultures were used as an external standard, there was a parallel loss of nitrite reagent specificity associated with storage of dipsticks in uncapped vials (Fig 1, broken line). Two urines, one lost and one reported as contaminated (mixed growth with three organisms, including diphtheroids and lactobacilli), were excluded from the analysis. DISCUSSION
The dip and read urine test is a useful adjunct to and, in certain circumstances, a substitute for microscopic urinalysis.‘.3~6.8 Although the package insert states that the vial containing these dipsticks must be kept tightly closed, in the physician laboratories in our ED this is rarely done. We suspect that this may be the case in other institutions as well. Through a simple, two-part, blinded, and controlled trial, we determined that exposure to room temperature and humidity over at least 2 weeks does not appear to alter the internal consistency and reproducibility of eight out of nine 121
122
AMERICAN
JOURNAL OF EMERGENCY
DAYS OF EXPOSURE IN OPEN CONTAINERS
FIGURE 1: Effect of exposure on specificity of nitrate reagent. (solid line) percent of false positives over 1 to 15 days of exposure, using fresh dipsticks as internal standard. ----- (broken line) percent of false positives over 1 to 15 days of exposure, using urine cultures as external standard.
Chemstrip-9 reagents. The reagents not substantially altered by exposure are those that measure urinary leukocyte esterase, pH, protein, glucose, ketones, urobilinogen, bilirubin, and blood. In contrast to this, the specificity of the nitrite reagent is affected by exposure to room air in a cumulative fashion over time. By the end of a week of exposure, 40% (8 out of 20) of the nitrite tests gave false-positive readings when measured against fresh dipsticks. At the end of 2 weeks, 75% (15 out of 20) gave false positive readings (Fig 1, solid line). Overall, 97% of the discordance between fresh and exposed nitrite reagents was attributable to false positives. False negatives were seen in only two instances (3%). To confirm these findings, we identified “uninfected” urine collected from asymptomatic out-patients. Samples were screened by negative nitrite and leukocyte esterase tests, and subsequently cultured. This screening test was chosen because the combination of negative nitrite and negative leukocyte esterase has been shown to be an excellent predictor of fewer than lo5 organisms/ml on culture.’ Eighteen out of 20 urine cultures (one lost, one contaminated) grew fewer than lo5 organisms/ml. The threshold of lo5 organisms/ml was chosen because it is the standard against which the nitrite reagent is normally measured.1*2*4,9 At the end of a week of exposure to room air, 33% of the nitrite tests gave false-positive readings when measured against urine culture results. After 2 weeks had elapsed, 72% of the readings were falsely positive for a specificity of 28% (Fig 1, broken line). In many instances the nitrite portion of the dipstick had turned pink (positive) in the open container before coming in contact with the urine. Thus, the findings in the second part of the study support those in the first part, as summarized in Fig 1. Whether a fresh dipstick is used as an internal standard or a negative urine culture is used as an external standard, there is a parallel increase in false-positive nitrite tests over a 2-week period of exposure to room air. The manufacturer, in describing the performance characteristics of the nitrite reagent, states in the package insert
MEDICINE
W Volume 8, Number 2 H March 1990
that “the frequency of false-positive results in normal patients is negligible (less than I%).” This claim is supported by the observations of others who have found the nitrite test to be 99.5 to 100% specific.2*9 Because the nitrite reagent has such a high specificity when stored under optimal conditions, many physicians routinely order a culture on any nitrite-positive urine. However, this clinical strategy may be rendered cost-ineffective by the occurrence of large numbers of false positives caused by storage of dipsticks in open containers. Other potential consequences of false-positive nitrite readings include unnecessary treatment of patients with antibiotics while awaiting culture results, or mistakenly attributing the source of fever in an ill patient to a urinary tract infection, thus leaving the actual source undetected. To avoid these pitfalls, one must either perform a microscopic urinalysis on all clinically suspected urinary tract infections, ensure that the dipstick containers are kept tightly closed, or date all vials when opened and routinely discard those that are more than a few days old. The use of the nitrite test to detect the presence of coliform bacteria in urine is a modification of a reagent that is more than a century old. lo Normal urine contains nitrates that are reduced to nitrites by most urinary pathogens.” These nitrites react with an aromatic amine that couples with another compound to produce a red-pink azo dye.” We speculate that the explanation for the high incidence of false positives found in this study may be caused by facilitation of this reaction by water vapor in the air. Because of the small number of false negatives in the first part of the study, the second portion was designed to investigate only false-positive nitrite readings. By design, the elimination of positive urine cultures made it impossible to calculate sensitivity. However, false-negative nitrite tests leading to low sensitivity are known to be common2.9 and may be caused by infection by organisms that do not reduce nitrates (gram positive cocci),13 insufficient bladder incubation time for reduction to occur,i4 insufficient nitrates in the urine available for reduction,4 and large amounts of dietary ascorbate.4 The conclusions that can be drawn from this study are limited by at least two considerations. Conditions described as room temperature and humidity were not measured and may fluctuate greatly. Thus the useful shelf life of an open container of dipsticks might show substantial geographic and seasonal variation. In addition, these observations describe the performance characteristics of only the Chemstrip-9 dipstick. The findings do not necessarily apply to dipsticks of other manufacturers. CONCLUSIONS We conclude that the high specificity of the nitrite portion of the Chemstrip-9 dipstick declines rapidly and cumulatively over time when these reagent sticks are stored in uncapped containers exposed to room air. This is in contrast to the stability of the other eight reagents which showed good reproducibility and internal consistency over at least 15 days of exposure. The authors would like to acknowledge Constance Verutes for her assistance in the editing and preparation of this manuscript.
GALLAGHER, SCHWARTZ, AND WEINSTEIN n PERFORMANCE OF URINE DIPSTICKS
REFERENCES 1. Loo SYT, Scottolini AG, Luangphinith S, et al: Urine screening strategy employing dipstick analysis and selective culture: An evaluation. Am J Clin Pathol 1984:81:634-642 2. Wise KA, Sagert LA, Grammers GL: Urine leukocyte esterase and nitrite tests as an aid to predict urine culture results. Lab Med 1964;15:166-167 3. Shaw ST, Poon SY, Wong ET: “Routine urinalysis”-Is the dipstick enough? JAMA 1985;253:1596-1600 4. Kiel DP, Moskowitz MA. The urinalysis: A critical appraisal. Med Clin North Am 1987;71:607-624 5. Stamm WE. Interpretation of urine cultures. Clin Microbial Newslett 1983;5:15-17 6. Kusumi RK, Grover PU, Kunin CM. Rapid detection of pyuria by leukocyte esterase activity. JAMA 1981;245:1653-1655 7. Benham L, O’Kell RT: Urinalysis: Minimizing microscopy. Clin Chem 1982;28:1722-1723
123
8. Schuman GB, Greenberg NF: Usefulness of macroscopic urinalysis as a screening procedure. Am J Clin Pathol 1979;71:452-456 9. Lenke RR, Van Dorsten JP: The efficacy of the nitrite test and microscopic urinalysis in predicting urine culture results. Am J Obstet Gynecol 1981;140:427-429 10. Griess P: Notes on the paper of Weselsky 8 Benedikt, some Azo compounds. Ber Dtsch Chem Ges 1879;12:426 11. Weltmann 0: Method for the simple detection of urinary tract infections. Wien Med Wschr 1922;72:618 12. Fuchs T, Gutensohn G: Value and limitation of the nitrite test in diagnosis of pyelonephritis. Dtsch Med J 1967;10:343 13. Sacks TG, Abramson JH: Screening tests for bacteruria: A validity study. JAMA 1967;201:79-82 14. Czenvinski AW, Wilkerson RG, Merrill JA, et al: Further evaluation of the Griess test to detect significant bacteruria. Am J Obstet Gynecol 1971 ;110:677-681
Dip and read urinalysis is a laboratory test commonly performed by emergency physicians. Although the manufacturer states that the capped vials contolning the dipsticks must be closed immediately after removal of a strip, this recommendation may not be followed in a busy emergency department. In a simple, two-part, blinded, and controlled trial the authors found that the reagents for determining leukocyte esterase, pH, protein, glucose, ketones, urobilinogen, blllrubin, and blood showed good reproducibility when fresh dipstlcks were compared with dipsticks exposed to room temperature and humidity for 1 to 15 days. In contrast to this, the nitrite portion of the exposed dipsticks showed a rapid and cumulative loss of specificity over time. By the end of a week of exposure, one third of the nitrite tests gave false-positive readings. At the end of a second week, nearly three quarters gave false-positive readings for a specificity of only 28%. It is concluded that the nitrite reagent, in contrast to the other eight reagents on the Chemstrtp-g dipstick (Biodynamics, Indianapolis, IN), rapidly loses accuracy when stored in uncapped vials. (Am J Emerg Med lggO;g:121-123. 0 1990 by W.B. Saunders Company.)
Clinically efficient, cost-effective algorithms using dip and read urinalysis are dependent on dipstick accuracy.‘” Accuracy of reagent strips is contingent on proper storage of these strips according to the manufacturer’s recommendations. A survey of the three physician laboratories in our emergency department (ED) showed that these containers are rarely recapped after removal of a reagent strip. We designed a simple, blinded, controlled trial to determine if exposure of urine dipsticks to room temperature and humidity affects their accuracy over time. METHODS
The Chemstrip-9 (Biodynamics, Indianapolis, IN) is an inert plastic strip made up of a sequence of nine discrete reagent pads for the determination of urinary leukocytes, nitrites, pH, protein, glucose, ketones, urobilinogen, bilirubin, and blood. The strip is immersed in urine for 1 second and read at 1 minute according to a color chart. One hundred sticks are packed in an aluminum vial with a tightly fitting stopper impregnated with a desiccant. In the first part of the study, 20 dipsticks from each of 10 containers left open for 1 to 15 days were used to test 20 urines collected at random in the ED for a total of 200 trials (20 dipstick urinalyses per container x 10 containers = 200 From the *Emergency Medical Services, Ambulatory Care, Bronx Municipal Hospital Center and the tDepartment of Medicine. Albert Einstein College of Medicine, Bronx, NY. Manuscript received April 6, 1969; accepted April 25, 1969. Address reprint requests to Dr Gallagher: Jacobi Hospital, Rm 1 W22, Bronx Municipal Hospital Center, Pelham Parkway S and Eastchester Rd, Bronx, NY 10461. Key Words: Urinalysis, errors in dipstick urinalysis, falsepositive nitrate reagent test. 0 1990 by W.B. Saunders Company. 0735.6757/90/0602-0006$5.00/O
MD,*t EDWARD SCHWARTZ, MD,* ROSLYN S. WEINSTEIN, MED*
trials). Results differing by more than one color shade from the reading of a fresh (control) dipstick were recorded as discordant by an observer who had been blinded to the age of the exposed dipsticks. Based on the findings from the initial portion of the study (Fig 1, solid line), the effect of exposure on the nitrite reagent portion of the dipstick was reexamined, using urine culture results as an external standard. Twenty dipsticks from each of 10 containers left open for 1 to 15 days were used to test urine collected from 20 ambulatory asymptomatic out-patients (200 trials). No patients were taking rifampin, phenazopyridine, or ascorbate,4 which might affect the accuracy of the nitrite test. Only patients with negative nitrite and leukocyte esterase tests were in this part of the study. Urines were subsequently cultured by the hospital laboratory using standard techniques. The observer reading the dipsticks had been blinded to the number of days each container had been exposed to room temperature and humidity. According to the manufacturer’s recommendation, any pink color of the nitrite reagent was recorded as positive. Cultures were considered positive if the urine grew more than 100,000 organisms/mL.5 RESULTS
Eight out of nine reagents showed excellent reproducibility and internal consistency when fresh urine dipsticks were compared with those exposed to room temperature and humidity for 1 to 15 days. The increasing discordance between the fresh and exposed nitrite reagent strips that occurred in 66 out of 68 instances (97%), was associated with a progressive increase in the number of false positives over time (Fig 1, solid line). In the second part of the study, when urine cultures were used as an external standard, there was a parallel loss of nitrite reagent specificity associated with storage of dipsticks in uncapped vials (Fig 1, broken line). Two urines, one lost and one reported as contaminated (mixed growth with three organisms, including diphtheroids and lactobacilli), were excluded from the analysis. DISCUSSION
The dip and read urine test is a useful adjunct to and, in certain circumstances, a substitute for microscopic urinalysis.‘.3~6.8 Although the package insert states that the vial containing these dipsticks must be kept tightly closed, in the physician laboratories in our ED this is rarely done. We suspect that this may be the case in other institutions as well. Through a simple, two-part, blinded, and controlled trial, we determined that exposure to room temperature and humidity over at least 2 weeks does not appear to alter the internal consistency and reproducibility of eight out of nine 121
122
AMERICAN
JOURNAL OF EMERGENCY
DAYS OF EXPOSURE IN OPEN CONTAINERS
FIGURE 1: Effect of exposure on specificity of nitrate reagent. (solid line) percent of false positives over 1 to 15 days of exposure, using fresh dipsticks as internal standard. ----- (broken line) percent of false positives over 1 to 15 days of exposure, using urine cultures as external standard.
Chemstrip-9 reagents. The reagents not substantially altered by exposure are those that measure urinary leukocyte esterase, pH, protein, glucose, ketones, urobilinogen, bilirubin, and blood. In contrast to this, the specificity of the nitrite reagent is affected by exposure to room air in a cumulative fashion over time. By the end of a week of exposure, 40% (8 out of 20) of the nitrite tests gave false-positive readings when measured against fresh dipsticks. At the end of 2 weeks, 75% (15 out of 20) gave false positive readings (Fig 1, solid line). Overall, 97% of the discordance between fresh and exposed nitrite reagents was attributable to false positives. False negatives were seen in only two instances (3%). To confirm these findings, we identified “uninfected” urine collected from asymptomatic out-patients. Samples were screened by negative nitrite and leukocyte esterase tests, and subsequently cultured. This screening test was chosen because the combination of negative nitrite and negative leukocyte esterase has been shown to be an excellent predictor of fewer than lo5 organisms/ml on culture.’ Eighteen out of 20 urine cultures (one lost, one contaminated) grew fewer than lo5 organisms/ml. The threshold of lo5 organisms/ml was chosen because it is the standard against which the nitrite reagent is normally measured.1*2*4,9 At the end of a week of exposure to room air, 33% of the nitrite tests gave false-positive readings when measured against urine culture results. After 2 weeks had elapsed, 72% of the readings were falsely positive for a specificity of 28% (Fig 1, broken line). In many instances the nitrite portion of the dipstick had turned pink (positive) in the open container before coming in contact with the urine. Thus, the findings in the second part of the study support those in the first part, as summarized in Fig 1. Whether a fresh dipstick is used as an internal standard or a negative urine culture is used as an external standard, there is a parallel increase in false-positive nitrite tests over a 2-week period of exposure to room air. The manufacturer, in describing the performance characteristics of the nitrite reagent, states in the package insert
MEDICINE
W Volume 8, Number 2 H March 1990
that “the frequency of false-positive results in normal patients is negligible (less than I%).” This claim is supported by the observations of others who have found the nitrite test to be 99.5 to 100% specific.2*9 Because the nitrite reagent has such a high specificity when stored under optimal conditions, many physicians routinely order a culture on any nitrite-positive urine. However, this clinical strategy may be rendered cost-ineffective by the occurrence of large numbers of false positives caused by storage of dipsticks in open containers. Other potential consequences of false-positive nitrite readings include unnecessary treatment of patients with antibiotics while awaiting culture results, or mistakenly attributing the source of fever in an ill patient to a urinary tract infection, thus leaving the actual source undetected. To avoid these pitfalls, one must either perform a microscopic urinalysis on all clinically suspected urinary tract infections, ensure that the dipstick containers are kept tightly closed, or date all vials when opened and routinely discard those that are more than a few days old. The use of the nitrite test to detect the presence of coliform bacteria in urine is a modification of a reagent that is more than a century old. lo Normal urine contains nitrates that are reduced to nitrites by most urinary pathogens.” These nitrites react with an aromatic amine that couples with another compound to produce a red-pink azo dye.” We speculate that the explanation for the high incidence of false positives found in this study may be caused by facilitation of this reaction by water vapor in the air. Because of the small number of false negatives in the first part of the study, the second portion was designed to investigate only false-positive nitrite readings. By design, the elimination of positive urine cultures made it impossible to calculate sensitivity. However, false-negative nitrite tests leading to low sensitivity are known to be common2.9 and may be caused by infection by organisms that do not reduce nitrates (gram positive cocci),13 insufficient bladder incubation time for reduction to occur,i4 insufficient nitrates in the urine available for reduction,4 and large amounts of dietary ascorbate.4 The conclusions that can be drawn from this study are limited by at least two considerations. Conditions described as room temperature and humidity were not measured and may fluctuate greatly. Thus the useful shelf life of an open container of dipsticks might show substantial geographic and seasonal variation. In addition, these observations describe the performance characteristics of only the Chemstrip-9 dipstick. The findings do not necessarily apply to dipsticks of other manufacturers. CONCLUSIONS We conclude that the high specificity of the nitrite portion of the Chemstrip-9 dipstick declines rapidly and cumulatively over time when these reagent sticks are stored in uncapped containers exposed to room air. This is in contrast to the stability of the other eight reagents which showed good reproducibility and internal consistency over at least 15 days of exposure. The authors would like to acknowledge Constance Verutes for her assistance in the editing and preparation of this manuscript.
GALLAGHER, SCHWARTZ, AND WEINSTEIN n PERFORMANCE OF URINE DIPSTICKS
REFERENCES 1. Loo SYT, Scottolini AG, Luangphinith S, et al: Urine screening strategy employing dipstick analysis and selective culture: An evaluation. Am J Clin Pathol 1984:81:634-642 2. Wise KA, Sagert LA, Grammers GL: Urine leukocyte esterase and nitrite tests as an aid to predict urine culture results. Lab Med 1964;15:166-167 3. Shaw ST, Poon SY, Wong ET: “Routine urinalysis”-Is the dipstick enough? JAMA 1985;253:1596-1600 4. Kiel DP, Moskowitz MA. The urinalysis: A critical appraisal. Med Clin North Am 1987;71:607-624 5. Stamm WE. Interpretation of urine cultures. Clin Microbial Newslett 1983;5:15-17 6. Kusumi RK, Grover PU, Kunin CM. Rapid detection of pyuria by leukocyte esterase activity. JAMA 1981;245:1653-1655 7. Benham L, O’Kell RT: Urinalysis: Minimizing microscopy. Clin Chem 1982;28:1722-1723
123
8. Schuman GB, Greenberg NF: Usefulness of macroscopic urinalysis as a screening procedure. Am J Clin Pathol 1979;71:452-456 9. Lenke RR, Van Dorsten JP: The efficacy of the nitrite test and microscopic urinalysis in predicting urine culture results. Am J Obstet Gynecol 1981;140:427-429 10. Griess P: Notes on the paper of Weselsky 8 Benedikt, some Azo compounds. Ber Dtsch Chem Ges 1879;12:426 11. Weltmann 0: Method for the simple detection of urinary tract infections. Wien Med Wschr 1922;72:618 12. Fuchs T, Gutensohn G: Value and limitation of the nitrite test in diagnosis of pyelonephritis. Dtsch Med J 1967;10:343 13. Sacks TG, Abramson JH: Screening tests for bacteruria: A validity study. JAMA 1967;201:79-82 14. Czenvinski AW, Wilkerson RG, Merrill JA, et al: Further evaluation of the Griess test to detect significant bacteruria. Am J Obstet Gynecol 1971 ;110:677-681