Efficacy of an Enzyme-Linked Immunosorbent Assay for Detection of Urinary Tract Immunoglobulins for Diagnosis of Urinary Tract Infections

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p /92/ $02.00/0 Copyright ) 1992, American Society for Microbiology Vol. 30, No. 7 Efficacy of an Enzyme-Linked Immunosorbent Assay for Detection of Urinary Tract Immunoglobulins for Diagnosis of Urinary Tract Infections J. A. KELLOGG,`* J. P. MANZELLA,2 J. W. SEIPLE,1 S. J. FORTNA,3t J. W. COOK,3 AND J. S. LEVISKY4 Immunology Laboratory, 1 General Internal Medicine,3 York Hospital, York, Pennsylvania 17405, and Department of Behavioral Sciences, York College of Pennsylvania, York, Pennsylvania Department of Medicine, Division of Infectious Diseases, 2 and Division of Received 24 February 1992/Accepted 24 April 1992 Results of the Uristat test (Shield Diagnostics Ltd.), a novel enzyme-linked immunosorbent assay (ELISA) for detection of urine antibodies to seven common bacterial pathogens, were compared with results of urine culture, urinalysis, and clinical history to determine the usefulness of Uristat in the diagnosis of urinary tract infections (UTIs). Midstream, catheterized, and indwelling catheter urine specimens sent to the laboratory for culture were included in the study. Quantitative cultures were performed on both 5% sheep blood agar and eosin-methylene blue agar. Uristat ELISAs were performed according to the manufacturer's instructions. By using a Bacillus subtilis bioassay technique, antibacterial activity was detected in the urine of 236 (22.2%) of 1,061 patients. Probable, possible, or asymptomatic UTIs were diagnosed for 258 (24.3%) of the 1,061 patients. Of those infections, 219 (84.9%o) were caused by bacterial species whose antibodies were detectable by Uristat. Uristat's sensitivity and specificity were 76.7 and 56.0%o, respectively. Uristat's predictive values of positive and negative results were 31.2 and 90.2%, respectively. Further development of the Uristat test is necessary before it can be of assistance in the diagnosis of UTIs. Clinical diagnosis of a urinary tract infection (UTI) is frequently difficult, particularly for women with frequency of urination and dysuria (29, 33, 39). In addition to a UTI, other causes of these symptoms include vulvovaginitis (with or without urethritis), genitourinary trauma, urethral irritants, and allergic reactions (9, 16). The diagnosis of a UTI can be made with greater accuracy in some patient populations if laboratory tests, including culture and microscopic determination of pyuria, hematuria, and bacteriuria, are performed (29, 33). However, none of these assays consistently provides a high level of both sensitivity and specificity. Culture results may be falsely positive because of contamination at the time of specimen collection from organisms in the distal urethra, vagina, and labial or periurethral skin (37). Culture results may also be falsely negative, or colony counts of isolates may be significantly reduced, because of factors including, but not limited to, the type or stage of disease, the use of antibiotics, the hydration of the patient, the specimen collection and transport methods used, and the type of pathogen involved (14, 16, 45). Measuring pyuria is a relatively simple, readily available means of establishing the presence of tissue invasion or the host's response to infection (37). Pyuria is a usually highly sensitive but sometimes nonspecific indicator of UTI (10, 11, 16, 18, 22, 29, 30, 32, 33, 37-39, 44). In contrast, microscopic determinations of hematuria and bacteriuria are highly specific for UTI but may (10) or may not (30, 44) lack sensitivity. The measurement of urine antibody levels may provide an alternative marker of host response to infection for use either as a screen or to assist, along with other tests, in establishing a diagnosis (22). Many of the antibodies in urine are locally produced in the urinary tract (1, 5, 7, 8, 12, 22, * Corresponding author. t Present address: The Miriam Hospital, Providence, RI ). Although patients with pyelonephritis appear to have the highest levels of urine antibody (1, 7, 8, 28, 31, 41, 43), those with cystitis (1, 4, 7, 22, 31, 34, 41, 43) and even asymptomatic infections (7, 8, 23) frequently have detectable antibodies. Lower levels of urine antibodies in bladder infections may be due to a reduced local immune response (41). Normal (uninfected) patients usually have no urine antibodies or very low levels of antibodies (7, 8, 12, 22, 28, 31, 34, 43). Antibodies detectable in the urine include secretory immunoglobulin A (IgA), IgA, and IgG (1, 4, 5, 7, 8, 12, 22, 28, 34, 40, 43) but usually do not include IgM (8, 12, 22, 34, 40). Peak urine antibody levels are usually reached within 10 days after the onset of infection during the period of symptoms (1, 4, 5). The levels may decline in many (but not all) patients, especially those with pyelonephritis, shortly after the cultures become negative (1, 5, 31). The purpose of the current study was to evaluate the diagnostic usefulness of the Uristat test (Shield Diagnostics Ltd., Dundee, Scotland). This indirect enzyme-linked immunosorbent assay (ELISA) permits detection of immunoglobulins in the urine of patients infected with any of the following seven uropathogens: Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Enterococcus faecalis. Wells on Uristat microtiter strips are coated with antigens isolated from these seven species. Although not yet approved for sale in the United States by the Food and Drug Administration, the Uristat test has been commercially available in Europe for more than 1 year. The ELISA is a very suitable means for measuring low levels of secretory IgA in urine (5, 7, 8, 34, 41). However, a single predetermined level of quantitative recovery of bacteria in culture cannot be used as a unilateral "gold standard" indicator of significant bacteriuria because of a lack of sensitivity (14, 25). Therefore, Uristat ELISA results were

2 1712 KELLOGG ET AL. compared in the current study not only with those of urine culture but also with results of urinalyses as well as with the clinical histories of the patients in order to determine the correlation of the Uristat results with the clinical diagnosis of infection. MATERIALS AND METHODS Patients and specimens. Randomly selected male and female patients from whom urine was submitted to the Clinical Microbiology Laboratory for culture were included in the study. Cultures were usually ordered either as part of a routine clinical workup or because of symptoms of UTI or sepsis. The patients were either hospitalized or seen by emergency department, hospital clinic, nursing home, or private physicians. Urine specimens were collected in sterile containers and sent to the laboratory on ice for urinalysis and either on ice or in urine transport kits (Becton Dickinson, Rutherford, N.J.) (17, 27) for both culture and urine antibody detection. Specimens received on ice were stored at 4 to 8 C and those in urine transport kits were stored at 25 C prior to laboratory analysis. Culture and urinalysis were performed within 4 h of specimen collection, and the Uristat ELISA was performed within 24 to 48 h of specimen collection. Urine cultures. Quantitative cultures were performed by using a ml loop to inoculate a tryptic soy agar plate with 5% defibrinated sheep blood and a 0.01-ml loop to inoculate an eosin-methylene blue agar plate. Colony counts were determined after the cultures were incubated at 35 C for 24 or 48 h. Bacterial and yeast isolates were identified to the species level by conventional techniques as previously described (15). Counts of.100 CFU (from eosin-methylene blue agar) or > 1,000 CFU (from sheep blood agar) of known urinary tract pathogens per ml of urine were considered potentially significant by the laboratory and were reported (14, 25). Determination of antibacterial activity in urine (26). A Mueller-Hinton agar plate was inoculated with 108 CFU of Bacillus subtilis ATCC 6633 per ml. After the inoculum had dried, 20,ul of mixed urine was added to a blank 6-mmdiameter paper disc on the plate, and the culture was incubated for 18 to 24 h at 35 C. Any zone of growth inhibition around the disc indicated the presence of antibacterial activity in the urine. Uristat ELISA detection of urine antibodies. The ELISA was performed as described by the manufacturer, except that all specimens were tested in duplicate to check for reproducibility of results. From each urine specimen, 100,ul was added to each of two Uristat test wells located on two different test strips. After 100 pul of wash buffer (as a blank), low-positive controls, and high-positive controls was pipetted into the respective wells, the test strips were incubated for 30 min at 25 C. The wells were decanted and washed three times with wash buffer, and then 100 [li of conjugate was added to each well. After incubation for another 30 min at 25 C, the wells were washed again three times, and 100,ul of substrate was added to each well. After incubation of the J. CLIN. MICROBIOL. strips for 15 min at 25 C, 100,ul of stop solution was added to the wells and the optical density of each well was determined at 554 nm. A patient's result was considered positive if the ratio of the result for the first of two wells tested per specimen to the result for the low-positive control was >1.0. Urinalysis. Urine specimens submitted for culture were analyzed by standard methods for the presence of erythrocytcs, protein, hemoglobin, and bacteria. Pyuria was determined by counting the number of leukocytes per high-power (magnification, x400) field from a centrifuged urine specimen. A result of.4 leukocytes per high-power field was considered positive (39) and appeared to offer, on the basis of our experience and that of previous reports (19, 39), satisfactory clinical correlation. Diagnosis of UTIs. The clinical history of each patient was reviewed by a physician participating in this study. Signs or symptoms including dysuria, urgency or frequency of urination, nocturia, flank pain, fever, hematuria, and a foul odor were all documented. In addition, a history of recent sexually transmitted disease or UTI, as well as recent administration of antibiotics (within 6 days of urine collection) or immunosuppressive drugs, was noted. The physicians' reason for ordering urine cultures was documented as either a routine workup (usually on hospital admission), evidence of sepsis (fever), or symptoms of a UTI. The diagnosis of UTI was based on previously determined criteria (25, 37). Patients were concluded either to be uninfected or to have a probable, possible, or asymptomatic infection. Patients considered to be uninfected had no signs or symptoms and < 100,000 CFU/ml of urine or were colonized with > 100,000 CFU/ml of urine but had no symptoms and no pyuria (37). Patients were considered to have a probable infection if they had at least one symptom and a culture containing a probable uropathogen at.100 CFU/ml. Those with a possible infection also had at least one symptom and a positive culture at >100 CFU/ml, but an alternative diagnosis could not be ruled out. Patients with asymptomatic infections had no specific symptoms but had both pyuria and a urine culture with >100,000 CFU of a uropathogen per ml. Physicians determining the clinical histories of patients were unaware of laboratory results from the most recent urine specimens from those patients. In addition, technologists performed either the Uristat ELISAs, culture, or urinalysis and were unaware of any other laboratory results from specimens that they were testing. The Z test for differences in proportions for independent samples was used for statistical analysis of results (2). Directional (one-tail) results were considered to be significant if the probability of the null hypothesis was 0.05 or less. Confidence intervals around the single-point proportion estimates were calculated by equation 1 of Simon (35). RESULTS From 18 March until 7 October 1991, urine specimens from 1,083 patients were processed. Seven patients were deleted from the study because of incomplete laboratory or clinical information. Uristat results for 15 additional patients were considered indeterminate because the infection status of the patients could not be determined. Of the remaining 1,061 patients, 304 (28.7%) were male and 757 (71.3%) were female. Patients ranged in age from 1 month to 95 years, with a mean of 47.7 years and a median of 46.0 years. Of the 1,061 specimens, 94 (8.8%) were indwelling catheter, 220 (20.7%) were catheterized, and 747 (70.4%) were midstream urine samples. Antibacterial activity in urine specimens. There was evidence of recent antimicrobial therapy, either from patient histories or from the B. subtilis bioassay, for 282 (26.6%) of the 1,061 specimens. A total of 233 (22.0%) of the patients had a history indicating recent use of antimicrobial agents. Such usage was confirmed by bioassay for 187 (80.2%) of these patients. Of the urine specimens tested, 236 (22.2%)

3 VOL. 30, 1992 IMMUNOLOGICAL DIAGNOSIS OF URINARY TRACT INFECTIONS 1713 TABLE 1. Uristat ELISA performance in detection of antibodies from patients' urine specimens Uristat result (%) Patient or culture No. of Uristat- Predictive value of: parameter (n) detectable infectionsa Sensitivity Specificity Positive Negative Sex Male (304) Female (757) result result Patient age in yr s1(2) (14) (29) (791) >70 (225) Culture reason Septic (96) Routine (425) Symptomatic (509) Other (31) Specimen collection method Indwelling catheter (94) Catheter (220) Midstream (747) Specimen transport method On ice Urine transport kit Unknown Antibiotics indicated from history and/or bioassay Present (282) Absent (779) Total patients (1,061) a Of 258 infections, 219 (84.9%) were caused by bacterial species whose antibodies were detectable by Uristat. had a bioassay result indicating the presence of antibacterial activity, including 44 (46.8%) of 94 indwelling catheter specimens, 48 (21.8%) of 220 catheterized specimens, and 144 (19.3%) of 747 midstream specimens. Recent usage of antimicrobial agents was confirmed by the clinical history for 187 (79.2%) of the 236 specimens with positive bioassay results. UTIs and Uristat performance. Probable, possible, or asymptomatic infections were diagnosed for 258 (24.3%) of the patients. Of these infections, 219 (84.9%) were due to pathogens whose antibodies were detectable by Uristat. Of these 219 infections, 168 were true positives and 51 were false negatives by Uristat. There were also 370 false-positive and 472 true-negative Uristat results. The overall Uristat sensitivity for these antibodies was 76.7% (confidence interval, to 81.82%); the sensitivity was 72.1% for female patients and 93.6% for males (P < 0.001) (Table 1). Uristat's sensitivity for antibodies was fairly low for the most part, regardless of the method of collection or transport of the specimen or the presence of antibiotics as determined by history, bioassay, or both. Uristat's sensitivity was, however, greater for patients who appeared septic than for those who had symptoms of a UTI (P < 0.01) or whose urine was routinely cultured (P < 0.05). Uristat's sensitivity varied significantly by type of infection (asymptomatic versus probable [P < 0.05]) but not by colony count of uropathogens (Table 2). Uristat's specificity and predictive value of a positive result were extremely low (56.0 and 31.2%, respectively), while the predictive value of a negative result was 90.2%. Of 51 specimens with false-negative Uristat results (as determined with the first of two duplicate test wells), none was positive in the second test well. Of the 1,061 patients, 10 (0.9%) had evidence of urinary tract colonization with bacteria. Uristat results were positive for seven (70%) of these patients. The reproducibility of Uristat results was excellent, with variation between the duplicate results usually being less than 3% and rarely exceeding 5%. DISCUSSION The Uristat test, in its current form, appears to be insufficiently sensitive or specific to aid routinely in the diagnosis of UTIs. Of the 258 infections diagnosed in the current study, only 219 (84.9%) were caused by bacterial species whose antibodies were detectable by this ELISA. The Uristat procedure detected only 168 (76.7%) of these infec-

4 1714 KELLOGG ET AL. TABLE 2. Sensitivity of Uristat as a function of classification and colony count of UTIs Infection type or No. of Uristat- Uristat result detectable colony count infections" No. of true Sensitivity Infection type Asymptomatic Probable 148 l Possible Colony count of bacteria (CFU/ml) > 100, , , ,001-10, < 1, Total infections " Of 258 infections, 219 (84.9%) were caused by bacterial species whose antibodies were detectable by Uristat. tions and only 65.1% of the total infections. Uropathogens which were encountered in this study and whose antibodies were not detectable by Uristat included Candida and Enterobacter species, Gardnerella vaginalis, and Staphylococcus epidermidis. The Uristat assay also cannot detect antibodies to Chlamydia trachomatis, another significant uropathogen (6), or to additional pathogens including Mycoplasma species, Corynebacterium species, and anaerobes (which are rarely uropathogens) (33, 45). The results of the current study, showing low Uristat sensitivity and specificity, are similar to those of a previous report of another commercially available ELISA system for detection of urinary tract antibodies (20). Falsely negative results of the Uristat test could be expected to occur because of many factors other than test insensitivity. These factors include decreased synthesis or increased degradation of antibodies in some patients (4, 20, 28, 34), variations in water diuresis (4, 21), antibiotic therapy (28), low levels of antibodies in infants younger than 1 year of age (1, 4, 8, 12), and the presence of either bacteria (1, 8) or soluble bacterial antigen (31) in the urine, resulting in absorption and a reduction in free detectable antibodies. Falsely positive Uristat results might also be expected for reasons other than lack of test specificity. Such reasons include leakage of serum antibodies through inflammed urinary tract mucosae (22, 28), prior immunologic stimulation by either normal intestinal flora or infection at another body site (28), prolonged elevation of antibody levels from previous UTIs (28), and recent insertion of catheters without bacteriuria (21). Uristat results may also appear falsely positive but may come from patients with false-negative cultures. Such situations may occur with patients on antibiotics (14, 20, 30, 38, 45) or those whose urine has inherent antibacterial activity (13, 14, 23, 24). Recent antibiotic use was indicated by patient history or bioassay for a high number (282 [27%]) of the patients whose urine specimens were submitted for culture in the current study. Our results showed that 80% of patients with a recent history of treatment with antimicrobial agents had positive bioassay results, while 79% of patients with positive bioassay results had a recent history of antibiotic administration. These results are very similar to those in a previous report (3). In the current study, however, the presence of antibacterial activity had no significant influence on the sensitivity of Uristat. Antibacterial activity in urine specimens may be due to factors other than antibiotic usage, including the presence of detergents (30), low ph (23, 24), high concentrations of urea (13, 23) and ammonium ions (13), increased osmolality (13, 23, 24), and the presence of prostatic fluid (36). In summary, careful documentation of symptoms, physical findings, and laboratory results are essential for the diagnosis and management of UTIs in many types of patients (33). 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