- Email: [email protected]
Evaluation of a rapid latex-particle agglutination-inhibition screening assay for cocaine in urine
670
Clinical and laboratory observations
ure. They also found a beneficial drop in the pulmonary/ systemic blood flow ratio in response to captopril when the systemic vascular resistance was high in the baseline state. Our data confirm the findings of this study and indicate that enalapriiat is a suitable intravenous pharmacologic probe of the renin-angiotensin system that may be reliably studied during diagnostic cardiac catheterization. Frenneaux et al.ll reported a beneficial response in clinical symptoms of eight infants with severe congestive heart failure when they were given enalapril orally. Six of their patients had large left-to-right shunts, and surgical therapy was significantly delayed or even avoided in four infants. Converting enzyme inhibition appears most useful in those infants with large left-to-right shunts and elevated systemic vascular resistance. Response to therapy may be tested by intravenous administration of enalaprilat during diagnostic cardiac catheterization, or oral angiotensin converting enzyme inhibitors could be used empirically in the treatment of infants with large left-to-right shunts and elevated systemic vascular resistance, in the hope of improving symptoms and possibly delaying or avoiding surgical therapy in selected patients.
REFERENCES 1. Berman W, Yabek S, Dillon T, Niland C, Corlew S, Christenson D. Effects of digoxin in infants with a congested circulatory state due to a ventricular septal defect. N Engl J Med 1983;308:363-6. 2. Alpert B, Barfield J, Taylor W. Reappraisal of digitalis in infants with left-to-right shunts and heart failure. J PEDIATR 1985;106:66-7. 3. White RD, Lietman PS. Commentary: a reappraisal of digitalis with left-to-right shunts and "heart failure." J PEDIATR 1978;92:867-70.
The Journal of Pediatrics October 1990
4. Baylen BG, Johnson G, Tsang R, Srivastava L, Kaplan S. The occurrence of hyperaldosteronism in infants with congestive heart failure. J Cardiol 1980;45:305-10. 5. Beckman RH, Rocchini AP, Rosenthal A. Hemodynamic effects of nitroprusside in infants with a large ventricular septal defect. Circulation 1981;64:553-8. 6. Cody R, Covit A, Schaer, G, Laragh J. Evaluation of a longacting converting enzyme inhibitor (enalapril) for the treatment of congestive heart failure. J Am Coil Cardiol 1983;1:1154-9. 7. DiCarlo L, Chatterjee K, Parmley W, et al. Enalapril, a new angiotensin converting enzyme inhibitor in chronic heart failure, acute and chronic hemodynamic evaluations. J Am Coil Cardiol 1983;2:865-71. 8. Gavras H, Faxon D, Berkoben J, Brunner H, Ryan T. Angiotensin converting enzyme inhibition in patients with congestive heart failure. Circulation 1978;58:770-6. 9. Kubo SH, Cody R J, Laragh J, et al. Immediate converting enzyme inhibition with intravenous enalapril in chronic congestive heart failure. Am J Cardiol 1985;55:122-6. 10. Shaddy RE, Teitel DF, Brett C. Short-term hemodynamic effects of captopril in infants with congestive heart failure. Am J Dis Child 1988;142:100-5. 11. Frenneaux M, Stewart R, Newman C, Hallidie-Smith K. Enalapril for severe heart failure in infancy. Arch Dis Child 1989;64:219-23. 12. Goll HM, Nyhan DP, Geller HS, Murray PA. Pulmonary vascular responses to angiotensin II and captopril in conscious dogs. J Appl Physiol 1986;61:1552-9. 13. LaFarge C, Miettinen O. The estimation of oxygen consumption. Cardiovasc Res 1970;4:23-30. 14. Kostis J. Angiotensin converting enzyme inhibitors. I. Pharmacology. Am Heart J 1980;116:1580-90. 15. Boucek M, Chang R. Effects of captopril on the distribution of left ventricular output with ventricular septal defect. Pediatr Res 1988;24:499-503.
Evaluation of a rapid latex-particle agglutination-inhibition screening assay for cocaine in urine Richard H. Schwartz, MD, Stuart Bogerna, PhD, a n d Mary M a r g a r e t Thorne, MT From Fairfax Hospital, Fails Church, Virginia, and American Medical Laboratories, Fairfax, Virginia
Submitted for publication March 16, 1990; accepted June 4, 1990. Reprint requests: Richard H. Schwartz, MD, 410 Maple Ave. West, Vienna, VA 22180. 9/28/22876
Several kits are now available for performing on-site screening of urine specimens for drugs of abuse. These kits employ a variety of techniques, including paper chromatography, 1-3 enzyme immunoassay, and latex agglutination-
Volume 117 Number 4
NIDA GC-MS
Clinical and laboratory observations
National Institute on Drug Abuse Gas chromatography-mass spectrometry
I
]
I
inhibition. Rapid tests for drugs of abuse could be of value in physicians' offices located in areas of high illicit drug use and in adolescent medicine clinics, emergency medicine facilities, prenatal clinics and intensive care nurseries, and pediatric intensive care units. We report the results of our analysis of the sensitivity, specificity, and accuracy of a latex-particle agglutinationinhibition test for cocaine metabolite. METHODS The Abuscreen OnTrak test kit (Roche Diagnostic Systems, Inc., Montclair, N.J.) is based on the latex agglutination-inhibition immunoassay technique. It yields a "yes" or "no" result; the color-change endpoint is simpler to interpret than paper chromatography methods. The kit is said by the manufacturer to provide the accuracy of immunoassay without the need for expensive equipment. The test for cocaine is also said to be specific for the major cocaine metabolite, benzoylecgonine. Adulterants such as table salt or alkaline or acidic substances will interfere with the results only when urinary concentrations of the drug metabolite approximate the threshold of detection (Salvatore Salamone, of Roche Diagnostics: personal communication, Sept. 12, 1989). All necessary materials, as well as detailed instructions, are contained in the kit. A box of 40 tests with a 4-month expiration date costs $120. The kit consists of a plastic slide containing an oval depression that is the mixing well, a mazelike track, and a viewing chamber. Reagents require refrigeration for storage but must be allowed to come to room temperature for 45 minutes before the test is performed. A single drop of urine, followed by the instillation of one drop each of three reagents (an antibody reagent, a buffer, and a latex reagent that is conjugated to a specific drug metabolite), is needed for each analysis. The urine-reagent mixture is gently stirred and pushed into the track entrance with the stirrer. The mixture moves along the track of the plastic-shielded slide by means of capillary action. Results are noted in a viewing chamber located at the end of the track. The test kit relies on the competition for binding to antibody between the latex-drug conjugate and the presence of a metabolite of a specific drug of abuse that may be present in the urine specimen. When the drug is present, it successfully competes with the latex-drug conjugate for the limited amount of antibody present. When the amount of drug metabolite in the specimen exceeds the cutoff point of the test, the metabolite in the specimen prevents the agglutination of latex particles. Thus a positive (drug-containing)
67 1
Table. Results of latex agglutination-inhibition
screening tests for cocaine metabolites GC-MS result
Cocaine test result
Positive
Negative
Total
Positive Negative TOTAL
35 2 37
1 15 16
36 17 53
result is indicated by a smooth, homogeneous milky appearance of the mixture of urine and reagents in the viewing chamber, tn the absence of drug in the urine, the latexdrug conjugate forms large, easily recognized agglutinated particles by binding to the antibody. The cutoff point (lowest reliably detected concentration of drug metabolite) for cocaine metabolites is 300 ng/ml. Results are obtained in approximately 3 minutes. As with all screening tests, analytic results are only preliminary, and a more specific method, preferably gas chromatography-mass spectrometry, must be used to obtain a confirmed result. Thirty-seven specimens presumptively positive by immunoassay and confirmed by GC-MS as positive for cocaine metabolites were interspersed by random code with 16 drug-free specimens. The GC-MS procedure included selected ion monitoring of three ions for each compound of interest and appropriate ion ratio for identification. The GC-MS procedures were performed according the U.S. Department of Health and Human Services National Institute on Drug Abuse guidelines in an NIDA-certified laboratory. The span of concentration of benzoylecgonine, the major urinary metabolite of cocaine as determined by GC-MS, was 116 to 530,000 ng/ml. Two specimens contained less than 300 ng/ml, the currently recommended N I D A workplace screening test cutoff point for cocaine metabolites. For analysis by the OnTrak kit, the specimens were marked only by a code number and positive and negative specimens were randomly and irregularly interspersed. Specimens had been frozen at - 6 0 ~ C for a period not exceeding 6 weeks. Most specimens were analyzed within 1 or 2 weeks after collection. Following the manufacturer's written instructions, a registered laboratory technologist with extensive experience in screening for drugs of abuse analyzed the specimens for cocaine metabolites with an OnTrak test kit. Results were recorded and mailed to one of us (R.H.S.), who had no prior knowledge of the results of the GC-MS tests. Results of GC-MS were compared with those of the OnTrak kit. RESULTS Of the 53 urine specimens, 37 were positive for cocaine metabolites by enzyme immunoassay screening procedure and confirmed as positive by GC-MS. The sensitivity of the
672
Clinical and laboratory observations
OnTrak test for cocaine metabolites was 95% and specificity was 94%. The predictive value of a positive result of a test for cocaine metabolites was 97%, and the predictive value ofa negatiwtest result was 89% (Table). The OnTrak test failed to detect one specimen that contained 116 ng/ml of benzoylecgonine as determined by GC-MS. Because the specimen contained cocaine metabolites at a concentration lower than the 300 ng/ml cutoff point for screening purposes, this result is not actually falsely negative. The result of a second test was indeterminate in comparison with the negative control supplied with the kit. We chose to record the indeterminate result as a negative result, in keeping with the manufacturer's recommendation. The other indeterminate result obtained with the OnTrak test kit contained 140,000 ng/ml of benzoylecgonine by GCMS. That urine specimen did not have any discernible characteristics, such as aberrant pH, color, odor, or specific gravity. There was one false positive result for cocaine metabolites. The laboratory technologist who performed the OnTrak tests stated that the test was easy to perform and the results were easy to recognize. In addition, the test was easily set up for rapid batch testing. DISCUSSION The Abuscreen OnTrak latex agglutination-inhibition test for detection of cocaine metabolites represents a new generation of on-site screening tests for drugs of abuse in urine. The test procedure is uncomplicated, rapid, and in-
The Journal of Pediatrics October 1990
expensive. The cost for materials needed for an individual drug test is $3 each for any of the five drug kits marketed by the manufacturer. Currently there are kits for the detection of marijuana, amphetamine, opiates, and barbiturate drug metabolites, in addition to the cocaine detection kit. The procedure is simple to perform, and the endpoint is clear for a test based on latex agglutination. The results for single drug testing are available in 3 minutes. The kit may be of particular value in drug abuse treatment facilities as an objective method of monitoring abstinence, such as after return from a weekend or overnight leave and during the period of posttreatment follow-up, when relapse can be expected to occur for 25% to 40% or more of those who complete inpatient treatment for cocaine dependency. Confirmation of positive immunoassay test results by a more specific analytic method for analysis of drugs of abuse preferably GC-MS, is strongly recommended. REFERENCES
1. Cone EJ, Menchen SL. Lack of validity of the KDI Quik Test drug screen for the detection of benzoylecgoninein urine. J Anal Toxicol 1988;11:276-7. 2. Bogema S, Schwartz RH, Godwin I. Evaluation of the Keystone Diagnostics Quik Test using previously screened urine specimens. J Anal Toxicol 1988;12:272-3. 3. Schwartz RH, Bogema S, Thorne MM. Evaluation of the Keystone Diagnostic Quik Test: a paper chromatography test of drugs of abuse in urine. Arch Pathol Lab Med 1989;113: 363-4.
Clinical and laboratory observations
ure. They also found a beneficial drop in the pulmonary/ systemic blood flow ratio in response to captopril when the systemic vascular resistance was high in the baseline state. Our data confirm the findings of this study and indicate that enalapriiat is a suitable intravenous pharmacologic probe of the renin-angiotensin system that may be reliably studied during diagnostic cardiac catheterization. Frenneaux et al.ll reported a beneficial response in clinical symptoms of eight infants with severe congestive heart failure when they were given enalapril orally. Six of their patients had large left-to-right shunts, and surgical therapy was significantly delayed or even avoided in four infants. Converting enzyme inhibition appears most useful in those infants with large left-to-right shunts and elevated systemic vascular resistance. Response to therapy may be tested by intravenous administration of enalaprilat during diagnostic cardiac catheterization, or oral angiotensin converting enzyme inhibitors could be used empirically in the treatment of infants with large left-to-right shunts and elevated systemic vascular resistance, in the hope of improving symptoms and possibly delaying or avoiding surgical therapy in selected patients.
REFERENCES 1. Berman W, Yabek S, Dillon T, Niland C, Corlew S, Christenson D. Effects of digoxin in infants with a congested circulatory state due to a ventricular septal defect. N Engl J Med 1983;308:363-6. 2. Alpert B, Barfield J, Taylor W. Reappraisal of digitalis in infants with left-to-right shunts and heart failure. J PEDIATR 1985;106:66-7. 3. White RD, Lietman PS. Commentary: a reappraisal of digitalis with left-to-right shunts and "heart failure." J PEDIATR 1978;92:867-70.
The Journal of Pediatrics October 1990
4. Baylen BG, Johnson G, Tsang R, Srivastava L, Kaplan S. The occurrence of hyperaldosteronism in infants with congestive heart failure. J Cardiol 1980;45:305-10. 5. Beckman RH, Rocchini AP, Rosenthal A. Hemodynamic effects of nitroprusside in infants with a large ventricular septal defect. Circulation 1981;64:553-8. 6. Cody R, Covit A, Schaer, G, Laragh J. Evaluation of a longacting converting enzyme inhibitor (enalapril) for the treatment of congestive heart failure. J Am Coil Cardiol 1983;1:1154-9. 7. DiCarlo L, Chatterjee K, Parmley W, et al. Enalapril, a new angiotensin converting enzyme inhibitor in chronic heart failure, acute and chronic hemodynamic evaluations. J Am Coil Cardiol 1983;2:865-71. 8. Gavras H, Faxon D, Berkoben J, Brunner H, Ryan T. Angiotensin converting enzyme inhibition in patients with congestive heart failure. Circulation 1978;58:770-6. 9. Kubo SH, Cody R J, Laragh J, et al. Immediate converting enzyme inhibition with intravenous enalapril in chronic congestive heart failure. Am J Cardiol 1985;55:122-6. 10. Shaddy RE, Teitel DF, Brett C. Short-term hemodynamic effects of captopril in infants with congestive heart failure. Am J Dis Child 1988;142:100-5. 11. Frenneaux M, Stewart R, Newman C, Hallidie-Smith K. Enalapril for severe heart failure in infancy. Arch Dis Child 1989;64:219-23. 12. Goll HM, Nyhan DP, Geller HS, Murray PA. Pulmonary vascular responses to angiotensin II and captopril in conscious dogs. J Appl Physiol 1986;61:1552-9. 13. LaFarge C, Miettinen O. The estimation of oxygen consumption. Cardiovasc Res 1970;4:23-30. 14. Kostis J. Angiotensin converting enzyme inhibitors. I. Pharmacology. Am Heart J 1980;116:1580-90. 15. Boucek M, Chang R. Effects of captopril on the distribution of left ventricular output with ventricular septal defect. Pediatr Res 1988;24:499-503.
Evaluation of a rapid latex-particle agglutination-inhibition screening assay for cocaine in urine Richard H. Schwartz, MD, Stuart Bogerna, PhD, a n d Mary M a r g a r e t Thorne, MT From Fairfax Hospital, Fails Church, Virginia, and American Medical Laboratories, Fairfax, Virginia
Submitted for publication March 16, 1990; accepted June 4, 1990. Reprint requests: Richard H. Schwartz, MD, 410 Maple Ave. West, Vienna, VA 22180. 9/28/22876
Several kits are now available for performing on-site screening of urine specimens for drugs of abuse. These kits employ a variety of techniques, including paper chromatography, 1-3 enzyme immunoassay, and latex agglutination-
Volume 117 Number 4
NIDA GC-MS
Clinical and laboratory observations
National Institute on Drug Abuse Gas chromatography-mass spectrometry
I
]
I
inhibition. Rapid tests for drugs of abuse could be of value in physicians' offices located in areas of high illicit drug use and in adolescent medicine clinics, emergency medicine facilities, prenatal clinics and intensive care nurseries, and pediatric intensive care units. We report the results of our analysis of the sensitivity, specificity, and accuracy of a latex-particle agglutinationinhibition test for cocaine metabolite. METHODS The Abuscreen OnTrak test kit (Roche Diagnostic Systems, Inc., Montclair, N.J.) is based on the latex agglutination-inhibition immunoassay technique. It yields a "yes" or "no" result; the color-change endpoint is simpler to interpret than paper chromatography methods. The kit is said by the manufacturer to provide the accuracy of immunoassay without the need for expensive equipment. The test for cocaine is also said to be specific for the major cocaine metabolite, benzoylecgonine. Adulterants such as table salt or alkaline or acidic substances will interfere with the results only when urinary concentrations of the drug metabolite approximate the threshold of detection (Salvatore Salamone, of Roche Diagnostics: personal communication, Sept. 12, 1989). All necessary materials, as well as detailed instructions, are contained in the kit. A box of 40 tests with a 4-month expiration date costs $120. The kit consists of a plastic slide containing an oval depression that is the mixing well, a mazelike track, and a viewing chamber. Reagents require refrigeration for storage but must be allowed to come to room temperature for 45 minutes before the test is performed. A single drop of urine, followed by the instillation of one drop each of three reagents (an antibody reagent, a buffer, and a latex reagent that is conjugated to a specific drug metabolite), is needed for each analysis. The urine-reagent mixture is gently stirred and pushed into the track entrance with the stirrer. The mixture moves along the track of the plastic-shielded slide by means of capillary action. Results are noted in a viewing chamber located at the end of the track. The test kit relies on the competition for binding to antibody between the latex-drug conjugate and the presence of a metabolite of a specific drug of abuse that may be present in the urine specimen. When the drug is present, it successfully competes with the latex-drug conjugate for the limited amount of antibody present. When the amount of drug metabolite in the specimen exceeds the cutoff point of the test, the metabolite in the specimen prevents the agglutination of latex particles. Thus a positive (drug-containing)
67 1
Table. Results of latex agglutination-inhibition
screening tests for cocaine metabolites GC-MS result
Cocaine test result
Positive
Negative
Total
Positive Negative TOTAL
35 2 37
1 15 16
36 17 53
result is indicated by a smooth, homogeneous milky appearance of the mixture of urine and reagents in the viewing chamber, tn the absence of drug in the urine, the latexdrug conjugate forms large, easily recognized agglutinated particles by binding to the antibody. The cutoff point (lowest reliably detected concentration of drug metabolite) for cocaine metabolites is 300 ng/ml. Results are obtained in approximately 3 minutes. As with all screening tests, analytic results are only preliminary, and a more specific method, preferably gas chromatography-mass spectrometry, must be used to obtain a confirmed result. Thirty-seven specimens presumptively positive by immunoassay and confirmed by GC-MS as positive for cocaine metabolites were interspersed by random code with 16 drug-free specimens. The GC-MS procedure included selected ion monitoring of three ions for each compound of interest and appropriate ion ratio for identification. The GC-MS procedures were performed according the U.S. Department of Health and Human Services National Institute on Drug Abuse guidelines in an NIDA-certified laboratory. The span of concentration of benzoylecgonine, the major urinary metabolite of cocaine as determined by GC-MS, was 116 to 530,000 ng/ml. Two specimens contained less than 300 ng/ml, the currently recommended N I D A workplace screening test cutoff point for cocaine metabolites. For analysis by the OnTrak kit, the specimens were marked only by a code number and positive and negative specimens were randomly and irregularly interspersed. Specimens had been frozen at - 6 0 ~ C for a period not exceeding 6 weeks. Most specimens were analyzed within 1 or 2 weeks after collection. Following the manufacturer's written instructions, a registered laboratory technologist with extensive experience in screening for drugs of abuse analyzed the specimens for cocaine metabolites with an OnTrak test kit. Results were recorded and mailed to one of us (R.H.S.), who had no prior knowledge of the results of the GC-MS tests. Results of GC-MS were compared with those of the OnTrak kit. RESULTS Of the 53 urine specimens, 37 were positive for cocaine metabolites by enzyme immunoassay screening procedure and confirmed as positive by GC-MS. The sensitivity of the
672
Clinical and laboratory observations
OnTrak test for cocaine metabolites was 95% and specificity was 94%. The predictive value of a positive result of a test for cocaine metabolites was 97%, and the predictive value ofa negatiwtest result was 89% (Table). The OnTrak test failed to detect one specimen that contained 116 ng/ml of benzoylecgonine as determined by GC-MS. Because the specimen contained cocaine metabolites at a concentration lower than the 300 ng/ml cutoff point for screening purposes, this result is not actually falsely negative. The result of a second test was indeterminate in comparison with the negative control supplied with the kit. We chose to record the indeterminate result as a negative result, in keeping with the manufacturer's recommendation. The other indeterminate result obtained with the OnTrak test kit contained 140,000 ng/ml of benzoylecgonine by GCMS. That urine specimen did not have any discernible characteristics, such as aberrant pH, color, odor, or specific gravity. There was one false positive result for cocaine metabolites. The laboratory technologist who performed the OnTrak tests stated that the test was easy to perform and the results were easy to recognize. In addition, the test was easily set up for rapid batch testing. DISCUSSION The Abuscreen OnTrak latex agglutination-inhibition test for detection of cocaine metabolites represents a new generation of on-site screening tests for drugs of abuse in urine. The test procedure is uncomplicated, rapid, and in-
The Journal of Pediatrics October 1990
expensive. The cost for materials needed for an individual drug test is $3 each for any of the five drug kits marketed by the manufacturer. Currently there are kits for the detection of marijuana, amphetamine, opiates, and barbiturate drug metabolites, in addition to the cocaine detection kit. The procedure is simple to perform, and the endpoint is clear for a test based on latex agglutination. The results for single drug testing are available in 3 minutes. The kit may be of particular value in drug abuse treatment facilities as an objective method of monitoring abstinence, such as after return from a weekend or overnight leave and during the period of posttreatment follow-up, when relapse can be expected to occur for 25% to 40% or more of those who complete inpatient treatment for cocaine dependency. Confirmation of positive immunoassay test results by a more specific analytic method for analysis of drugs of abuse preferably GC-MS, is strongly recommended. REFERENCES
1. Cone EJ, Menchen SL. Lack of validity of the KDI Quik Test drug screen for the detection of benzoylecgoninein urine. J Anal Toxicol 1988;11:276-7. 2. Bogema S, Schwartz RH, Godwin I. Evaluation of the Keystone Diagnostics Quik Test using previously screened urine specimens. J Anal Toxicol 1988;12:272-3. 3. Schwartz RH, Bogema S, Thorne MM. Evaluation of the Keystone Diagnostic Quik Test: a paper chromatography test of drugs of abuse in urine. Arch Pathol Lab Med 1989;113: 363-4.