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Stability of Legionella urinary antigens over time
ELSEVIER
BACTERIOLOGY
Stability of Legionella Antigens Over Time
Urinary
E.W. Rigby, J.F. Plouffe, B.A. Hackman, D.S. Hill, R.F. Benson, and R.F. Breiman
Twenty-two urine samples positive for Legionella pneumophila serogroup 1 antigen by EQUATE radioimmunoassay (RlA) (Binax, Portland, ME, USA) were stored at various temperatures and the RIA repeated at 1, 7, 30, 90, and 120 days to evaluate stability of the urina y antigens. The mean
INTRODUCTION Untreated Legionnaires’ disease has a mortality rate as high as 25%. The etiologic agent of 50 to 60% of all cases of Legionnaires’ disease is Legion&r pneumophiZu serogroup 1 (Reingold et al. 1984). Various methods can be employed for the diagnosis of Legionnaires’ disease. The Legionella urinary antigen assay (Binax, Portland, ME) is a useful diagnostic tool, because it is rapid, specific, and sensitive (Kohler et al. 1981; Aguero-Rosenfeld et al. 1988; Ruf et al. 1990; Plouffe et al. 1995; Dominguez et al. 1996). While comparing the new enzyme-linked immunosorbent assay (ELISA) to previously positive samples by radioimmunoassay (RIA) that had been frozen for up to 3 years, it was discovered that the ratios had decreased from 18.0 to 4.6 (p = .002X Because many hospitals do not routinely test for Legionella antigen, but send samples to outside labs, we were concerned
From the Ohio State University Medical Center, Columbus, Ohio (EWR, JFP, BAH, DSH), Centers for Disease Control and Prevention (RF Benson, RF Breiman), Atlanta, Georgia, USA. Address reprint requests to Joseph F. Plouffe, M.D., N-1135 Doan Hall, 410 W. 10th Avenue, Columbus, Ohio 43210, USA. Presented at the 96th General Meeting, American Society for Microbiology, New Orleans, May 1996. Received 27 September 1996; revised and accepted 16 December 1996.
DIAGN MICROBIOL INFECT DIS 1997;28:1-3 0 1997 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
ratios of patient/negative control remained stable. Although there was a 10% decrease in the mean ratios after 1 month, changes were not significant. However, individual samples with ratios close to 3 may fall to 43. 0 1997 Elsevier Science Inc.
about the stability of the urinary antigen over time and at various storage temperatures.
MATERIALS
AND METHODS
Twenty-two patients with pneumonia with positive assays for Legionella urinary antigen using the Equate RIA Legionella Urinary Antigen Kit (Binax) were studied. Assays were performed according to manufacturer’s directions. A urine sample from each patient was pipetted into a tube precoated with polyclonal rabbit antibody specific for L. pneumophilu serogroup 1 antigen. After incubation for 1 hour, the tube was aspirated and washed to remove any remaining unbound sample. I125 labeled polyclonal rabbit antibody was then added to the tubes, which were again incubated and rinsed. Bound radioactivity was measured as counts per minute (cpm) on a gamma counter. A sample was considered positive when the cpm were greater than or equal to three times the activity of the negative controls. The 22 positive urine samples were divided into aliquots in 13 x 100 mm plastic tubes. One aliquot was stored at room temperature (24°C) and another at 4°C. The remaining aliquots were stored at -20°C. The assay was repeated on samples stored at each temperature after 1, 7, and 30 days. In addition, the
0732~8893/97/$17.00 PII SO732-8893(97)00019-9
E.W. Rigby et al.
2
TABLE
1 Mean Ratio Values + Standard Deviation
over a l- and
6-Month Period Storage Temp
Grouv
Initial Value
Loti
24”C, 1 Month
4”C, 1-6 Months
-2O”C, 1-6 Months
3.95 + 0.66
3.91 ? 1.06
3.86 * 0.74 5.74 k 4.80
3.60 f 0.58 5.04 c 3.08
Midb
10.38 f 4.59
7.83 + 3.60
9.19 * 4.02 10.66 * 7.15
8.79 + 5.14 11.22 + 8.62
High’
42.38 + 9.07
40.86 + 7.00
36.38 + 8.58 39.92 c 11.16
35.12 + 10.29 34.08 + 8.68
“Ratio between 3 and 5 bRatio >5 to ~20 ‘Ratio >20
assay was repeated on samples stored at 4°C and -20°C after 90 and 120 days. The ratios were compared to day 0 results using the paired t-test.
RESULTS The initial Legionella urinary antigen ratios from the 22 patients ranged from 3.1 to 53.7. Samples were grouped by ratio as low positive (ratio = 3 to 5, N = 8), midrange (ratios > 5 to 20, N = 9), and high (ratio > 20, N = 5). After 1, 7, and 30 days, the mean ratios did not decrease significantly (Table 1). However, one sample became negative both at room temperature and at 4°C after 30 days, with ratios declining from 3.5 to 2.4 and 2.6, respectively. The mean ratios of the samples stored for 3 and 6 months at 4°C and -20°C decreased, but not significantly. Three samples in the low group became negative (ratio < 3) at some point in time. One of the nine midrange samples became negative at 4°C after 6 months (1.6) and another at -20°C at 6 months (2.8) (see Table 2).
TABLE
2
Number
Positive/Number
DISCUSSION The Legionella urinary antigen test is useful for rapid diagnosis of Legionnaires’ disease. The test has not been used widely partly because of the need for radioisotopes. Recently an ELISA has become commercially available through Binax that had similar sensitivity and specificity (Hackman et al. 1996). Chang et al. (19961, however, demonstrated that the ELISA kit was more sensitive than the RIA in detecting positive samples in specimens frozen at -70°C for up to 1 year. While we were comparing the ELISA and RIA kits on samples that had been frozen for up to 3 years, we noted that ratios had decreased significantly. It is important to know at what time and under which storage conditions the antigen begins to lose stability. Some laboratories batch samples until there are enough to perform the assay. Others send samples to reference laboratories. Still other research laboratories store the urine samples for future testing. Mean Legionella urinary antigen ratios were stable for at least 1 month after being stored at 25”, 4”, and -20°C. Although there were no significant differences among means, individual urine samples with
Tested (%) over a l- and 6-Month Period Storage Temp 4”C,
-2O”C,
Grouv
24”C, 1 Month
1 Month
6 Months
1 Month
6 Months
Low” Midb High’
7/8 (88) 8/9 (89) 5/5 (100)
7/8 (88) 9/9 (100) 5/5 (100)
6/S (75) B/9 (89) 5/5 (100)
8/8 (100) 8/9 (78) 5/5 (100)
6/8 (75) 7/9 (78) 5/5 (100)
“Ratio between 3 and 5 bRatio >5 to ~20 ‘Ratio >20
Stability
of Legionella
urinary antigens
initial ratios between 3 and 5 occasionally had ratios less than 3 on retesting. Approximately one-third of our positive samples fell into the low-positive group. This prospective experiment was only carried out for 6 months, and we did not see the decrease in ratios previously noted after 3 years of storage. Occasional samples with ratios between 3 and 5 had ratios ~3 after storage. We feel that ratios >2.5 should be viewed as highly suggestive of Legionnaires’ disease. Legionella antigens in urine are relatively stable
3
and can be stored and shipped at various temperatures without major changes in the results.
We thank the members of the CBPIS group: Ian Baird, Jill Porterfield-Baxa, Jane Emerick, Robert J Fuss, George Gianakopoulos, Mark Herbert, Susan L. Koletar, William Maher, Janet A. Minor, Michael F. Para, James Parsons, and Cathy S. Tumbleson.
REFERENCES Aguero-Rosenfeld ME, Edelstein PH (1988) Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in human. ] Clin Microbial 26:17751778. Chang FY, Stout JE, Yu V (1996) Assessment of enzyme immunoassay versus radioimmunoassay for detection of Legionella pneumoph~la serogroup 1 antigen in frozen urine specimens. J Clin Microbial 34:2628-2629.
Kohler RB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, White A (1981) Rapid radioimmunoassay diagnosis of Legionnaires’ disease: Detection and partial characterization of urinary antigen. Ann Int Med 94:601-605. Plouffe JF, File TM, Breiman R, Hackman BA, Salstrom SJ, Marston BJ, Fields BS, the Community-Based Pneumonia Incidence Study Group (1995) Re-evaluation of the definition of Legionnaires’ disease: Use of the urinary antigen assay. Clin Infect Dis 20:1286-91.
Dominguez JA, Manterola JM, Blavia R, Sopena N, Belda FJ, Padilla E, Gimenez M, Sabria M, Morera J, Ausina V (1996) Detection of Legionella pneumophila serogroup 1 antigen in nonconcentrated urine and urine concentrated by selective ultrafiltration. I Clin Microbial 34:2334-2336.
Reingold AL, Thomason RM, Brake BJ, Thacker L, Wilkinson HW, Kuritsky JN (1984) Legionella pneumonia in the United States: The distribution of serogroups and species causing human illness. J Infect Dis 149819.
Hackman BA, Plouffe JF, Benson RF, Fields BS, Benson RF (1996) Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen. J Clin Microbial 34:1579-1580.
Ruf B, Schurmann D, Horback I, Fehrenbach FJ, Pohle HD (1990) Prevalence and diagnosis of Legionella pneumonia: A 3-year prospective study with emphasis on application of urinary antigen detection. I Infect Dis 162:1341-1348.
BACTERIOLOGY
Stability of Legionella Antigens Over Time
Urinary
E.W. Rigby, J.F. Plouffe, B.A. Hackman, D.S. Hill, R.F. Benson, and R.F. Breiman
Twenty-two urine samples positive for Legionella pneumophila serogroup 1 antigen by EQUATE radioimmunoassay (RlA) (Binax, Portland, ME, USA) were stored at various temperatures and the RIA repeated at 1, 7, 30, 90, and 120 days to evaluate stability of the urina y antigens. The mean
INTRODUCTION Untreated Legionnaires’ disease has a mortality rate as high as 25%. The etiologic agent of 50 to 60% of all cases of Legionnaires’ disease is Legion&r pneumophiZu serogroup 1 (Reingold et al. 1984). Various methods can be employed for the diagnosis of Legionnaires’ disease. The Legionella urinary antigen assay (Binax, Portland, ME) is a useful diagnostic tool, because it is rapid, specific, and sensitive (Kohler et al. 1981; Aguero-Rosenfeld et al. 1988; Ruf et al. 1990; Plouffe et al. 1995; Dominguez et al. 1996). While comparing the new enzyme-linked immunosorbent assay (ELISA) to previously positive samples by radioimmunoassay (RIA) that had been frozen for up to 3 years, it was discovered that the ratios had decreased from 18.0 to 4.6 (p = .002X Because many hospitals do not routinely test for Legionella antigen, but send samples to outside labs, we were concerned
From the Ohio State University Medical Center, Columbus, Ohio (EWR, JFP, BAH, DSH), Centers for Disease Control and Prevention (RF Benson, RF Breiman), Atlanta, Georgia, USA. Address reprint requests to Joseph F. Plouffe, M.D., N-1135 Doan Hall, 410 W. 10th Avenue, Columbus, Ohio 43210, USA. Presented at the 96th General Meeting, American Society for Microbiology, New Orleans, May 1996. Received 27 September 1996; revised and accepted 16 December 1996.
DIAGN MICROBIOL INFECT DIS 1997;28:1-3 0 1997 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
ratios of patient/negative control remained stable. Although there was a 10% decrease in the mean ratios after 1 month, changes were not significant. However, individual samples with ratios close to 3 may fall to 43. 0 1997 Elsevier Science Inc.
about the stability of the urinary antigen over time and at various storage temperatures.
MATERIALS
AND METHODS
Twenty-two patients with pneumonia with positive assays for Legionella urinary antigen using the Equate RIA Legionella Urinary Antigen Kit (Binax) were studied. Assays were performed according to manufacturer’s directions. A urine sample from each patient was pipetted into a tube precoated with polyclonal rabbit antibody specific for L. pneumophilu serogroup 1 antigen. After incubation for 1 hour, the tube was aspirated and washed to remove any remaining unbound sample. I125 labeled polyclonal rabbit antibody was then added to the tubes, which were again incubated and rinsed. Bound radioactivity was measured as counts per minute (cpm) on a gamma counter. A sample was considered positive when the cpm were greater than or equal to three times the activity of the negative controls. The 22 positive urine samples were divided into aliquots in 13 x 100 mm plastic tubes. One aliquot was stored at room temperature (24°C) and another at 4°C. The remaining aliquots were stored at -20°C. The assay was repeated on samples stored at each temperature after 1, 7, and 30 days. In addition, the
0732~8893/97/$17.00 PII SO732-8893(97)00019-9
E.W. Rigby et al.
2
TABLE
1 Mean Ratio Values + Standard Deviation
over a l- and
6-Month Period Storage Temp
Grouv
Initial Value
Loti
24”C, 1 Month
4”C, 1-6 Months
-2O”C, 1-6 Months
3.95 + 0.66
3.91 ? 1.06
3.86 * 0.74 5.74 k 4.80
3.60 f 0.58 5.04 c 3.08
Midb
10.38 f 4.59
7.83 + 3.60
9.19 * 4.02 10.66 * 7.15
8.79 + 5.14 11.22 + 8.62
High’
42.38 + 9.07
40.86 + 7.00
36.38 + 8.58 39.92 c 11.16
35.12 + 10.29 34.08 + 8.68
“Ratio between 3 and 5 bRatio >5 to ~20 ‘Ratio >20
assay was repeated on samples stored at 4°C and -20°C after 90 and 120 days. The ratios were compared to day 0 results using the paired t-test.
RESULTS The initial Legionella urinary antigen ratios from the 22 patients ranged from 3.1 to 53.7. Samples were grouped by ratio as low positive (ratio = 3 to 5, N = 8), midrange (ratios > 5 to 20, N = 9), and high (ratio > 20, N = 5). After 1, 7, and 30 days, the mean ratios did not decrease significantly (Table 1). However, one sample became negative both at room temperature and at 4°C after 30 days, with ratios declining from 3.5 to 2.4 and 2.6, respectively. The mean ratios of the samples stored for 3 and 6 months at 4°C and -20°C decreased, but not significantly. Three samples in the low group became negative (ratio < 3) at some point in time. One of the nine midrange samples became negative at 4°C after 6 months (1.6) and another at -20°C at 6 months (2.8) (see Table 2).
TABLE
2
Number
Positive/Number
DISCUSSION The Legionella urinary antigen test is useful for rapid diagnosis of Legionnaires’ disease. The test has not been used widely partly because of the need for radioisotopes. Recently an ELISA has become commercially available through Binax that had similar sensitivity and specificity (Hackman et al. 1996). Chang et al. (19961, however, demonstrated that the ELISA kit was more sensitive than the RIA in detecting positive samples in specimens frozen at -70°C for up to 1 year. While we were comparing the ELISA and RIA kits on samples that had been frozen for up to 3 years, we noted that ratios had decreased significantly. It is important to know at what time and under which storage conditions the antigen begins to lose stability. Some laboratories batch samples until there are enough to perform the assay. Others send samples to reference laboratories. Still other research laboratories store the urine samples for future testing. Mean Legionella urinary antigen ratios were stable for at least 1 month after being stored at 25”, 4”, and -20°C. Although there were no significant differences among means, individual urine samples with
Tested (%) over a l- and 6-Month Period Storage Temp 4”C,
-2O”C,
Grouv
24”C, 1 Month
1 Month
6 Months
1 Month
6 Months
Low” Midb High’
7/8 (88) 8/9 (89) 5/5 (100)
7/8 (88) 9/9 (100) 5/5 (100)
6/S (75) B/9 (89) 5/5 (100)
8/8 (100) 8/9 (78) 5/5 (100)
6/8 (75) 7/9 (78) 5/5 (100)
“Ratio between 3 and 5 bRatio >5 to ~20 ‘Ratio >20
Stability
of Legionella
urinary antigens
initial ratios between 3 and 5 occasionally had ratios less than 3 on retesting. Approximately one-third of our positive samples fell into the low-positive group. This prospective experiment was only carried out for 6 months, and we did not see the decrease in ratios previously noted after 3 years of storage. Occasional samples with ratios between 3 and 5 had ratios ~3 after storage. We feel that ratios >2.5 should be viewed as highly suggestive of Legionnaires’ disease. Legionella antigens in urine are relatively stable
3
and can be stored and shipped at various temperatures without major changes in the results.
We thank the members of the CBPIS group: Ian Baird, Jill Porterfield-Baxa, Jane Emerick, Robert J Fuss, George Gianakopoulos, Mark Herbert, Susan L. Koletar, William Maher, Janet A. Minor, Michael F. Para, James Parsons, and Cathy S. Tumbleson.
REFERENCES Aguero-Rosenfeld ME, Edelstein PH (1988) Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in human. ] Clin Microbial 26:17751778. Chang FY, Stout JE, Yu V (1996) Assessment of enzyme immunoassay versus radioimmunoassay for detection of Legionella pneumoph~la serogroup 1 antigen in frozen urine specimens. J Clin Microbial 34:2628-2629.
Kohler RB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, White A (1981) Rapid radioimmunoassay diagnosis of Legionnaires’ disease: Detection and partial characterization of urinary antigen. Ann Int Med 94:601-605. Plouffe JF, File TM, Breiman R, Hackman BA, Salstrom SJ, Marston BJ, Fields BS, the Community-Based Pneumonia Incidence Study Group (1995) Re-evaluation of the definition of Legionnaires’ disease: Use of the urinary antigen assay. Clin Infect Dis 20:1286-91.
Dominguez JA, Manterola JM, Blavia R, Sopena N, Belda FJ, Padilla E, Gimenez M, Sabria M, Morera J, Ausina V (1996) Detection of Legionella pneumophila serogroup 1 antigen in nonconcentrated urine and urine concentrated by selective ultrafiltration. I Clin Microbial 34:2334-2336.
Reingold AL, Thomason RM, Brake BJ, Thacker L, Wilkinson HW, Kuritsky JN (1984) Legionella pneumonia in the United States: The distribution of serogroups and species causing human illness. J Infect Dis 149819.
Hackman BA, Plouffe JF, Benson RF, Fields BS, Benson RF (1996) Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen. J Clin Microbial 34:1579-1580.
Ruf B, Schurmann D, Horback I, Fehrenbach FJ, Pohle HD (1990) Prevalence and diagnosis of Legionella pneumonia: A 3-year prospective study with emphasis on application of urinary antigen detection. I Infect Dis 162:1341-1348.