- Email: [email protected]
A Test for Determining Significant Bacterial Levels in Urine
THE JOURNAL OF UROLOGY
Vol. 92, No. 6 December 1964 Copyright © 1964 by The Williams & Wilkins Co. Printed in U.S.A.
A TEST FOR DETERMINING SIGNIFICANT BACTERIAL LEVELS IN URINE LEWIS J. GRIFFITH
AND
ZANE F. SMITH
From the Laboratory Service, Veterans Administration Hospital, Batavia, New York
There has been increasing recognition that a large percentage of patients with active urinary infections are asymptomatic. Autopsies have confirrn.ed the development of severe renal damage in such individuals. 1 This knowledge has led to a greater emphasis on routine bacteriological testing of urine, even when symptorn.atology is lacking, and has emphasized the need for a simple, accurate, preliminary method of screening urinary tract infections. Although colony counting provides an accurate indication of bacterial levels, the inherent mechanics and complexity of this technique make it impractical for screening large groups routinely, or for urine tests in the doctor's office. It has been recognized that a sirn.ple test pennitting the doctor to detect infectious levels of bacteria easily and rapidly in his own office would be a valuable diagnostic aid for early detection of urinary infections. Simmons and 1Villiams2 have recently published studies reporting the use of triphenyltetrazolium chloride (T.T.C.) as an indicator in a simple test for significant levels of bacteria. The use of T.T.C. is based on observations by Wundt3 that actively metabolizing bacteria reduce soluble colorless T.T.C. to an insoluble red triphenyl formazan. Simmons and Williams designed their test to give positive results at levels of 100,000 bacteria or more per milliliter of urine after 4 hours of incubation at 37C, using uncatheterized specimens. Selection of 100,000 bacteria per milliliter as the dividing line bet,veen true urinary infection and chance contamination was based on the generally accepted conclusions of previous studies. 4 • 5 This Accepted for publication May 28, 1964. 1 Jackson, G. G., Dallenbach, F. D. and Kipnis, G. P.: Symposium on clinical advances in medicine; Pyelonephritis; correlation of clinical and pathological observations in the antibacterial era. Med. Clin. N. Amer., 39: 297, 1955. 2 Simmons, N. A. and Williams, J. D.: Simple test for significant bacteriuria. Lancet, 1: 1377, 1962. 3 Wundt, W.: Investigation of reduction action of bacteria on triphenyltetrazolium chloride. Deutsch. Med. Wchnschr., 75: 1471, 1950. 4 Kass, E. H.: Asymptomatic infections of the urinary tract. Tr. A. Am. Physicians, 69: 56, 1956.
eliminated the need for catheterization with its attendant discomfort and potential dangers. 6 Simmons and Williams used a liquid preparation of buffered T.T.C. Due to the limited stability of such solutions, it was necessary for fresh solutions to be prepared frequently. This restricted the use of the Simmons and Williams test. A stable, dry form of buffered T.T.C. would offer certain advantages and its recent availability stimulated a new study.* MATERIALS AND METHODS
A total of 856 urine specimens were used in this study. Uncatheterized specimens collected by midstream or clean catch techniques were used. Such specin1ens were used in conformity with the assumption that a large scale screening method could not be successfully based on catheterized specimens. The test was performed by adding 2 ml. of thoroughly mixed urine specimen to a test tube containing dried, buffered T.T.C. reagent. The specimen was mixed until the reagent dissolved and then incubated at 37C for 4 hours. The tests were examined at 2 and 4 hours. Parallel colony counts of each urine specimen were conducted using a 10-fold dilution technique. This permitted correlation between the colony counting method and the T.T.C. test after 2 and 4 hours' incubation. A negative result was identi fied by the absence of a colored precipitate. A positive result was identified by the appearance of a pink-to-red precipitate (triphenyl formazan). The T.T.C. test was performed on urine specimens even when they were highly colored or con-tained blood. By examining the sediment from the T.T.C. tube on a glass slide, the formazan precipitate was readily distinguished from red blood 5 Merritt, A. D. and Sanford, J. P.: Sterilevoided urine culture; evaluation in 100 consecutive hospitalized women. J. Lab. Clin. Med., 52: 463, 1958. 6 Brumfitt, W., Davies, B. I. and Rosser, E. I. Urethral catheter as a cause of urinary tract infection in pregnancy and puerperium. Lancet, 2: 1059-1062, 1961. * Uroscreen, a dried buffered T.T.C. reagent is supplied by Knickerbocker Biologics, Pfizer Laboratories, Chas. Pfizer & Co., Inc.
721
722 TABLE
GRIFFITH AND SJVIITH
1. Uroscreen clinical testing: Six separate
field investigations summarized
Total number of urine specimens Total number of false positives Total number of false negatives corrected for antibacterial therapy Corrected per cent of accuracy
856 58 42
Total number of urine specimens with 100,000 organisms per ml. or greater by culture Total number of false negatives corrected for antibacterial therapy Corrected per cent of accuracy on urine specimens with 100,000 organisms per ml. or greater
270
88%
42 84.5%
cells. An alternate procedure on such specimens was centrifuging and resuspending the sediment in distilled water, thus lysing the red blood cells, and in the case of highly colored urine, enhancing the readability of the test. RESULTS
The presence of glucose, ketones and urobilinogen was found to have no effect on test results. 2 When immediate testing was not possible, it was found that test results were not affected by storing urine specimens under refrigeration (4C) up to 24 hours. Refrigerated specimens show no significant change in viable bacteria. 7 A false positive has been classified as a urine specimen containing less than 100,000 organisms per milliliter and yielding a positive T.T.C. test reaction. Fiftyeight such specimens were found. Urine specimens containing more than 100,000 organisms per milliliter on colony count, but giving a negative result, were classified as false negatives. Patients known to be on antimicrobial therapy at the time of testing (total 15) were deleted from this group (table 1). DISCUSGION
The T.T.C. test provided an 85 to 92 per cent range of correlation with the bacterial colony counting method in individual reports. 7 Ryan, W. L. and Mills, R. D.: Bacterial multiplication in urine during refrigeration. Amer. J. Med. Tech., 29: 175-180, 1963.
Because the test is based on reduction of T.T.C. by actively metabolizing bacteria, the data indicated that results may be influenced by antimicrobial medication and that the T.T.C. test should be performed before antibacterial therapy is administered or after systemic antibacterial levels have resumed normalcy. The reported clinical investigation indicates the usefulness of the test for mass screening in connection with periodic physical or hospital admission examinations. The simplicity of this technique makes it useful for routine urine testing in the doctor's office. The only equipment required for the test is a 37C incubator and it is conveniently easy to perform. The dried, buffered T.T.C. test provides a rapid and simple means of testing large numbers of patients routinely for urinary infections and permits frequent testing of patient groups with high susceptibility to such infections (i.e. diabetes, hypertensives, pregnant women). 8 Additional testing of positive T.T.C. tubes indicated that one could use this material very successfully for gram staining, further culture and sensitivity testing. These are the normal procedures that would customarily follow an indication of significant bacterial levels in urine. It is believed that dried, buffered T.T.C. satisfactorily complements colony counting and other current techniques and that it offers a reliable preliminary method of screening for urinary infections. Acknowledgment of aid in completing this evaluation is given to Dr. Erwin Neter, Children's Hospital, Buffalo, New York; Dr. Henry Isenberg, Long Island Jewish Hospital; Dr. Howard Lind, Brooks Hospital, Brookline, Massachusetts; Capt. Kenneth A. Borchardt, Letterman General Hospital, San Francisco, California, and Dr. Gerald Needham, Mayo Clinic, Rochester, Minnesota. 8 Chard, C. and Cole, P. G.: Diagnosis of significant bacteriuria in pregnancy. Lancet, 2: 326-
327, 1963.
Vol. 92, No. 6 December 1964 Copyright © 1964 by The Williams & Wilkins Co. Printed in U.S.A.
A TEST FOR DETERMINING SIGNIFICANT BACTERIAL LEVELS IN URINE LEWIS J. GRIFFITH
AND
ZANE F. SMITH
From the Laboratory Service, Veterans Administration Hospital, Batavia, New York
There has been increasing recognition that a large percentage of patients with active urinary infections are asymptomatic. Autopsies have confirrn.ed the development of severe renal damage in such individuals. 1 This knowledge has led to a greater emphasis on routine bacteriological testing of urine, even when symptorn.atology is lacking, and has emphasized the need for a simple, accurate, preliminary method of screening urinary tract infections. Although colony counting provides an accurate indication of bacterial levels, the inherent mechanics and complexity of this technique make it impractical for screening large groups routinely, or for urine tests in the doctor's office. It has been recognized that a sirn.ple test pennitting the doctor to detect infectious levels of bacteria easily and rapidly in his own office would be a valuable diagnostic aid for early detection of urinary infections. Simmons and 1Villiams2 have recently published studies reporting the use of triphenyltetrazolium chloride (T.T.C.) as an indicator in a simple test for significant levels of bacteria. The use of T.T.C. is based on observations by Wundt3 that actively metabolizing bacteria reduce soluble colorless T.T.C. to an insoluble red triphenyl formazan. Simmons and Williams designed their test to give positive results at levels of 100,000 bacteria or more per milliliter of urine after 4 hours of incubation at 37C, using uncatheterized specimens. Selection of 100,000 bacteria per milliliter as the dividing line bet,veen true urinary infection and chance contamination was based on the generally accepted conclusions of previous studies. 4 • 5 This Accepted for publication May 28, 1964. 1 Jackson, G. G., Dallenbach, F. D. and Kipnis, G. P.: Symposium on clinical advances in medicine; Pyelonephritis; correlation of clinical and pathological observations in the antibacterial era. Med. Clin. N. Amer., 39: 297, 1955. 2 Simmons, N. A. and Williams, J. D.: Simple test for significant bacteriuria. Lancet, 1: 1377, 1962. 3 Wundt, W.: Investigation of reduction action of bacteria on triphenyltetrazolium chloride. Deutsch. Med. Wchnschr., 75: 1471, 1950. 4 Kass, E. H.: Asymptomatic infections of the urinary tract. Tr. A. Am. Physicians, 69: 56, 1956.
eliminated the need for catheterization with its attendant discomfort and potential dangers. 6 Simmons and Williams used a liquid preparation of buffered T.T.C. Due to the limited stability of such solutions, it was necessary for fresh solutions to be prepared frequently. This restricted the use of the Simmons and Williams test. A stable, dry form of buffered T.T.C. would offer certain advantages and its recent availability stimulated a new study.* MATERIALS AND METHODS
A total of 856 urine specimens were used in this study. Uncatheterized specimens collected by midstream or clean catch techniques were used. Such specin1ens were used in conformity with the assumption that a large scale screening method could not be successfully based on catheterized specimens. The test was performed by adding 2 ml. of thoroughly mixed urine specimen to a test tube containing dried, buffered T.T.C. reagent. The specimen was mixed until the reagent dissolved and then incubated at 37C for 4 hours. The tests were examined at 2 and 4 hours. Parallel colony counts of each urine specimen were conducted using a 10-fold dilution technique. This permitted correlation between the colony counting method and the T.T.C. test after 2 and 4 hours' incubation. A negative result was identi fied by the absence of a colored precipitate. A positive result was identified by the appearance of a pink-to-red precipitate (triphenyl formazan). The T.T.C. test was performed on urine specimens even when they were highly colored or con-tained blood. By examining the sediment from the T.T.C. tube on a glass slide, the formazan precipitate was readily distinguished from red blood 5 Merritt, A. D. and Sanford, J. P.: Sterilevoided urine culture; evaluation in 100 consecutive hospitalized women. J. Lab. Clin. Med., 52: 463, 1958. 6 Brumfitt, W., Davies, B. I. and Rosser, E. I. Urethral catheter as a cause of urinary tract infection in pregnancy and puerperium. Lancet, 2: 1059-1062, 1961. * Uroscreen, a dried buffered T.T.C. reagent is supplied by Knickerbocker Biologics, Pfizer Laboratories, Chas. Pfizer & Co., Inc.
721
722 TABLE
GRIFFITH AND SJVIITH
1. Uroscreen clinical testing: Six separate
field investigations summarized
Total number of urine specimens Total number of false positives Total number of false negatives corrected for antibacterial therapy Corrected per cent of accuracy
856 58 42
Total number of urine specimens with 100,000 organisms per ml. or greater by culture Total number of false negatives corrected for antibacterial therapy Corrected per cent of accuracy on urine specimens with 100,000 organisms per ml. or greater
270
88%
42 84.5%
cells. An alternate procedure on such specimens was centrifuging and resuspending the sediment in distilled water, thus lysing the red blood cells, and in the case of highly colored urine, enhancing the readability of the test. RESULTS
The presence of glucose, ketones and urobilinogen was found to have no effect on test results. 2 When immediate testing was not possible, it was found that test results were not affected by storing urine specimens under refrigeration (4C) up to 24 hours. Refrigerated specimens show no significant change in viable bacteria. 7 A false positive has been classified as a urine specimen containing less than 100,000 organisms per milliliter and yielding a positive T.T.C. test reaction. Fiftyeight such specimens were found. Urine specimens containing more than 100,000 organisms per milliliter on colony count, but giving a negative result, were classified as false negatives. Patients known to be on antimicrobial therapy at the time of testing (total 15) were deleted from this group (table 1). DISCUSGION
The T.T.C. test provided an 85 to 92 per cent range of correlation with the bacterial colony counting method in individual reports. 7 Ryan, W. L. and Mills, R. D.: Bacterial multiplication in urine during refrigeration. Amer. J. Med. Tech., 29: 175-180, 1963.
Because the test is based on reduction of T.T.C. by actively metabolizing bacteria, the data indicated that results may be influenced by antimicrobial medication and that the T.T.C. test should be performed before antibacterial therapy is administered or after systemic antibacterial levels have resumed normalcy. The reported clinical investigation indicates the usefulness of the test for mass screening in connection with periodic physical or hospital admission examinations. The simplicity of this technique makes it useful for routine urine testing in the doctor's office. The only equipment required for the test is a 37C incubator and it is conveniently easy to perform. The dried, buffered T.T.C. test provides a rapid and simple means of testing large numbers of patients routinely for urinary infections and permits frequent testing of patient groups with high susceptibility to such infections (i.e. diabetes, hypertensives, pregnant women). 8 Additional testing of positive T.T.C. tubes indicated that one could use this material very successfully for gram staining, further culture and sensitivity testing. These are the normal procedures that would customarily follow an indication of significant bacterial levels in urine. It is believed that dried, buffered T.T.C. satisfactorily complements colony counting and other current techniques and that it offers a reliable preliminary method of screening for urinary infections. Acknowledgment of aid in completing this evaluation is given to Dr. Erwin Neter, Children's Hospital, Buffalo, New York; Dr. Henry Isenberg, Long Island Jewish Hospital; Dr. Howard Lind, Brooks Hospital, Brookline, Massachusetts; Capt. Kenneth A. Borchardt, Letterman General Hospital, San Francisco, California, and Dr. Gerald Needham, Mayo Clinic, Rochester, Minnesota. 8 Chard, C. and Cole, P. G.: Diagnosis of significant bacteriuria in pregnancy. Lancet, 2: 326-
327, 1963.