bacteriuria. a screening method for Firefly luciferase ...

Firefly luciferase ATP assay as a screening method for bacteriuria. A Thore, A Lundin and S Anséhn J. Clin. Microbiol. 1983, 17(2):218.

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Vol. 17, No. 2

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1983, p. 218-224

0095-1137/83/020218-07$02.OO/O Copyright © 1983, American Society for Microbiology

Firefly Luciferase ATP Assay as a Screening Method for Bacteriuria A. THORE,lt* A. LUNDIN,1t AND S. ANStHN2 National Defence Research Institute, Department 4, S-100 64 Umea',l and Department of Clinical Bacteriology, Linkoping University Hospital, S-581 85 Linkoping,2 Sweden

Received 21 May 1982/Accepted 1 November 1982

Several authors have demonstrated the potential usefulness of the firefly luciferase assay of bacterial ATP as a method for rapid detection of bacteriuria (1, 4, 6, 8, 9, 12, 13). The method is based on the rough proportionality between bacterial ATP and numbers of CFU observed in bacterial cultures and, to some extent, verified in urine specimens (1, 4, 6, 8, 9, 12, 13). By using a discriminatory level of ATP corresponding to approximately 105 CFU/ml in the urine, it has been possible by luciferase analysis to achieve sensitivities ranging between 75 and 98% as compared with plate count techniques (Table 1). A major concern has been the specificity of the luciferase test. To remove nonbacterial ATP, urines are pretreated with the detergent Triton X-100 in the presence of the ATP-degrading enzyme apyrase (1, 4, 6, 8, 9, 12, 13). In model experiments, this treatment has been shown to selectively destroy ATP in a variety of somatic cells, leaving bacterial ATP more or less unaffected (1, 4, 12, 13). In urine specimens containing 105 CFU/ml were less affected (13). However, significant numbers of culture-negative specimens retain sufficient ATP, even after the treatment, for these samples to be classified as "luciferase positives" at a cutoff level of ATP corresponding to 105 CFU/ml. Attempts have been made to reduce the number of "false positives" by modifying the pretreatment. Modifications include pretreatment at elevated temperatures (6), or at acid pH (1, 4, 8, 12) followed by further acidification to extract bacterial ATP, and finally, by neutralization. However, although the positive effects of these modifications have been demonstrated in model experiments, their effects in clinical urine specimens are inconclusive, and numbers of false-positive specimens remain significant (Table 1). Therefore, since results with our previously described method (13) compare favorably with those of other, more laborious procedures, we decided to evaluate our method in a larger and betterdefined clinical material. A total of 2,018 randomly selected clinical urine specimens sent to a bacteriological laboratory were assayed, and the results were compared with culture results. MATERIALS AND METHODS Urine specimens. All urine specimens sent for routine culture and arriving before noon at the clinical 218


A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding >105 CFU/ml and specimens yielding 105 CFU/ml and 88% of specimens yielding 105 CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 105 to 106.

FIREFLY LUCIFERASE ATP ASSAY

VOL. 17, 1983

219

TABLE 1. Results with firefly luciferase bacteriuria test as compared with routine plate count Predictive Reference

(13)

Thore et al. 1975

(13)

No. of preincubation steps

(%

+

-

91/17 183/32

75 81

28 92

19 70

83 96

4 x 10-11 2 x 10-1o

4 4

319/94

98

59

35

99

4 x 10`0

2

319/94

91

83

55

98

6 x 10-9*

2

348/63

73

93

70

94

6x

10`1

3

957/207

87

85

57

97

Not statede

Flow system

538/124

97 85

74

46 47

99 96

Not stated 1 x 10-9*

6 Flow system

105 CFU/ml

1976c (9)

Gutekunst 1977 (8) Curtis and Johnston 1979c"d

1,653/220

78

(6) a Normalized to the average prevalence of culture-positive urines, i.e., 18.5%. b Values correspond to 105 bacterial per ml according to the respective authors, regardless of whether this value has been expressively used to define a limit for a positive test. Values marked with asterisks are "best-fit values" resulting in maximum difference between culture positives and negatives, suggested by the authors instead of values corresponding to 10- bacteria per ml. c CFU determined by pour plate method. d Specimens with equivocal plate counts (68) not included. Calibrated by 105 CFU/ml of a laboratory culture of Escherichia coli. bacteriological laboratory in Linkoping University hospital for a period of about 3 weeks were included in the study. A total of 2,018 specimens were analyzed. Most specimens were clean voided midstream samples. From the youngest children, the urine was collected in sterile adhesive plastic bags, and from elderly patients, many specimens were obtained via indwelling catheters. In Table 2 the distribution of urine specimens according to patient category is shown. Bacteriological procedures. Quantitative cultures were performed with calibrated loops on Endo agar (Difco Laboratories) and on blood agar. After incubation at 37°C for 16 to 20 h, the counts of CFU per milliliter were determined, and the microorganisms were classified, mainly according to Cowan and Steel (5). Since slow-growing and fastidious organisms

might be overlooked in routine culture, a random sample of 200 consecutive specimens was submitted to "extended culture" consisting of prolonged incubation for another 24 h on Endo- and blood agar plates. This sample was also cultured on Sabouraud dextrose agar (Oxoid Ltd.) and chocolate agar (BBL Microbiology Systems) and by anaerobic culture methods. An additional 5.5% positive cultures were obtained in this way (Table 3). In addition to the random sample, all specimens with a positive ATP assay yielding 105 CFU/ml on extended >10' CFU/ml Specimens Organism

Not identified Mixed flora Coliforms Proteus Pseudomonas

culture

culture Random sample (200)

Random sample (200)

False luciferase positives (201)

5 23 257 36 8 16 32 0

0 2 25 3 0 1 4 0

0 5 2 0 0 1 2 1

2 11 2 2 0 12 7 3

Staphylococci Streptococci Candida albicans

volume of 2 ml. An aliquot of 1 ml was then added to 4 specimens arrived at the laboratory, by use of the BM ml of boiling 0.1 M Tris buffer (pH 7.75) containing 2 Test-Nitrite (Boehringer Mannheim Corp.). mM EDTA and was incubated (100°C, 5 min) to extract bacterial ATP and to inactivate apyrase. RESULTS The extracts were kept refrigerated and were assayed within a few hours for bacterial ATP by the were divided into four specimens The 2,018 previously described firefly luciferase method (13). groups with respect to yields in routine culture: Measurement of bioluminescence was made in a home-built luminometer equipped with an RCA 1P21 106 CFU/ml. photomultiplier (13). ATP concentrations of the urine The percentages of luciferase positives in these specimens were calculated by using assays of a known groups as a function of the ATP limit defining a amount of ATP as reference, correcting for reagent positive luciferase test are shown in Fig. 1. Depending on the clinical purpose of the test blanks and the 10-fold dilution of the urine in the preparation of the extracts. (see below), several approaches can be used to Correction was also made for inhibition of the determine the ATP limit for a luciferase-positive luciferase reaction by urine constituents by multiplica- test. At a limit of 13.5 nM ATP, the difference in tion of the primary results by a factor of 2.7, which percentages of luciferase positives between the was found in a previous study to correspond to the average inhibition by urine under these conditions group with 104 to 105 CFU/ml and the group with (13). This is close to values which can be calculated i05 to 106 CFU/ml was at a maximum (Fig. 1). from a recent study on urine inhibition of the luciferase At this limit, the percentages of luciferase positives were 11% (105 CFU/ml)/(total number of luciferase or nitrite positives). b Expressed as (number of luciferase or nitrite negatives with 105 CFU/ml (35%) were classified as negative by the luciferase test. The distribution of the 45% false-negative nitrite tests was also random according to patient category, with the possible exception of the specimens from the maternal care unit, in which four of five of the specimens containing >105 CFU/ml (80%) were classified as nitrite negative. The distribution of false luciferase negatives and false nitrite negatives in relation to bacterial species is shown in Table 6. To allow for the possibility that the false-negative results were due to lack of sensitivity of the chemical methods, the occurrence of false-negative chemical tests in specimens yielding 10' to 106 CFU/ml was compared with that in specimens yielding >106 CFU/ml. It is evident that the majority of the falsenegative luciferase tests occurred in specimens with low culture yields, whereas most of the false-negative nitrite tests occurred in specimens with high yields (Table 6). The total number of false-negative luciferase tests was too low to allow an evaluation of the distribution among


(>106 CFU/ml). Due to the high number of specimens in the group with 105 CFU/ml had >67.5 nM ATP, and most specimens with 105 CFU/ml were negative. Sensitivities, specificities, and predictive values (7) for the luciferase and nitrite tests are summarized in Table 5. Routine culture with a cutoff limit at 10' CFU/ml was used as refer-

Measure

Nitrite test

Luciferase test (ATP level in urine) 13.5-67.5 nM

222

THORE, LUNDIN, AND ANSEHN

J. CLIN. MICROBIOL.

TABLE 6. Number of false luciferase negative and nitrite negative specimens in relation to routine culture yield and bacterial species No. of false-negative specimens" Luciferase assay Microorganism(s) Lcfrs sa Microorganism(s) Yield: >106 bacteria Yield: per ml 105_106 105_1O6

Nitrite test

>106 bacteria per ml

bacterial species, whereas the false-negative nitrite tests were clearly overrepresented in specimens yielding mixed flora, staphylococci, and streptococci. Most of the false luciferase negative specimens in the low culture yield category were also negative with the nitrite test (18 of 20). The influence on different bacterial species of varying the ATP limit for a positive luciferase test is shown in Fig. 2. The majority of specimens containing 105 to 106 CFU/ml of Proteus spp. (6 specimens) or Streptococcus spp. (16

(4) (18) (226) (29) (8) (6) (16)

specimens) remain positive even at a relatively high ATP limit. This is in contrast to results with other bacterial species and may indicate a higher number of cells per CFU in these species as compared with, e.g., coliforms or Staphylococcus spp.

False-positive specimens. The only significant deviation from a random distribution of the 12% false luciferase positive specimens (Table 5) among different patient categories was found in boys below the age of 14, from whom only 2 of

A. All species

B. Mixed flora

C. Gram negative sp.

D. Gram positive sp.

100-

(,,

50-

Cl)

7n 0

L-

z ..

UJ

---p---S V.-~~~~~~~~~~~~l

------

'L50 l ~ ~ ~~'__ ~

=

2 04dP LI 0

o -\0

do

0

1

do

ATP LIMIT FOR POSITIVE TEST (nM)

FIG. 2. Percentage of positive luciferase tests in routine culture specimens with yields of >105 CFU/ml when the ATP limit defining a positive test was varied. The culture groups were 105 to 106 CFU/ml (dotted lines) and >106 CFU/ml (solid lines). (A) All species: CFU >106 (O); CFU 105 to 106 (0). (B) Mixed flora (23 specimens): CFU >106/ml (0); CFU 105 to 106 (0). (C) Gram-negative species: coliforms (257 specimens) (0); Proteus spp. (35 specimens) (0). (D) Gram-positive species: Staphylococcus spp. (16 specimens) (0); Streptococcus spp. (32 specimens) (0). The number of specimens in each group is given in Table 6.


Not identified 1 (1) 1 (4) 1 (1) 0 Mixed flora 3 (5) 1 (18) 4 (5) 13 Coliformsb 9 (31) 7 (226) 20 (31) 68 Proteus 1 (6) 1 (29) 4 (6) 14 1 (1) Pseudomonas 1 (8) 1 (1) 3 2 (10) Staphylococci 0 (6) 10 (10) 3 3 (16) Streptococci 0 (16) 14 (16) 14 a Numbers in parentheses refer to total numbers of specimens yielding >105 CFU/ml in each group. b E. coli, Klebsiella spp., and other coliforms.

VOL. 17, 1983

DISCUSSION In the luciferase test for bacteriuria, bacterial ATP is measured after the removal of nonbacterial ATP by incubation with Triton X-100 and apyrase. Since ATP is a universal intracellular metabolite and is always present at similar levels in viable bacteria (12, 13), the luciferase test would be expected to be generally applicable, regardless of the type of bacteria or the patient's diet. This has been shown to be a problem with other chemical tests, e.g., the nitrite test (2), which is known to have high specificity but low sensitivity. The major problem in using the luciferase test is to define a concentration of ATP corresponding to a culture yielding -105 CFU/ml according to the criteria described by Kass (10). Estimations of the ATP content of laboratory cultures of bacterial strains vary widely according to different authors, in many cases probably due to methodological problems. However, cited estimations often are on the order of 10-18 to 10-17 mol of ATP per bacterial cell (12, 13). This is in agreement with a cutoff level corresponding to 105 CFU/ml at 4 x 10-1o M ATP (13). Limits in this range have also been used by several authors (6, 12, 13). Also, lower limits of 4 x 10-1l to 6 x 10-11 M ATP have been used (1, 4), based on lower estimations of the amount of ATP per CFU. This may be explained by experimental conditions in these experiments, which include filtration and incubation at low pH (1, 4), which would be expected to reduce bacterial ATP levels. Using the theoretically based cutoff levels of ATP, the luciferase test has resulted in sensitivities in excess of 70% compared with routine plate counts of >105 CFU/ml; in some investigations, sensitivities approach 100% (Table 1).

223

Thus, the major problem has been the specificity, which has varied between 28% (4) and 93% (1). The problem of specificity has been interpreted to reflect inefficient removal of nonbacterial ATP, and much effort has been used to optimize the Triton X-100 apyrase treatment (6, 12). Although model experiments have been encouraging, results with actual urine specimens seem to be little improved (Table 1). An alternative approach would be to consider the positioning of the cutoff ATP level. In this connection, it is worth noting that in actual urine specimens, the average ATP per CFU is considerably higher in specimens with low CFU levels than in those with heavy bacterial growth (13). From data in Fig. 6 of our previous work (13), the average ATP level in urines containing close to 105 CFU/ml can be calculated to be 5 x 10-9 M, i.e., more than 10-fold higher than the average based on the entire material. Although a contributing factor may have been failure to remove nonbacterial ATP, similarly high ratios of ATP per CFU have also been observed in laboratory cultures at low cell densities when cells were grown in the presence of urine or urea (3). The high ATP level corresponding to 105 CFU was confirmed in the present study, in which an ATP level of 13.5 nM was found to result in maximum differentiation between the group with 104 to 105 CFU/ml and the group with 105 to 106 CFU/ml (Fig. 1). Taking 13.5 nM ATP as the limit for a positive luciferase test, an overall sensitivity of 92% and a specificity of 88% were obtained compared with routine culture, in which 105 CFU was used as the discriminatory level. The 8% (31 specimens) of false negatives were found mainly in the group with 10 to 106 CFU/ml and were few in the group with >106 CFU/ml (Table 6). This was in contrast to the nitrite test, which gave a large number of false negatives in the >106 group. Of the 12% (201 specimens) false luciferase positives, 41 specimens turned out to yield >105 CFU/ml when subjected to an extended culture procedure (Table 3). The possibility of additional, noncultivatable bacteria in the material cannot be excluded. The clinical significance of the detected fastidious organisms is not clear; however, several potential pathogens were found among these false luciferase positives (Table 3). It should be noted that increasing the cutoff limit of ATP from 13.5 to 67.5 nM decreased the number of false luciferase positives from 12 to 3%, i.e., from 201 to 55 specimens, and increased the number of false luciferase negatives from 8 to 21%, i.e., from 31 to 67 specimens. At the limit of 67.5 nM ATP, the number of specimens correctly classified by the luciferase test was at a maximum. The decisive factor in evaluating any diagnos-


70 cultures containing 105 CFU/ml (Table 3), which is four times the expected frequency as found in the random material (Table 3). Most of the additional positive cultures yielded mixed flora, staphylococci, or streptococci. A high proportion (9 of 20) of the false nitrite positive specimens were also false luciferase positive.

FIREFLY LUCIFERASE ATP ASSAY

224

THORE, LUNDIN, AND ANStHN

LITERATURE CITED 1. Alexander, D. N., G. M. Ederer, and J. M. Matsen. 1976. Evaluation of an adenosine 5'-triphosphate assay as a screening method to detect significant bacteriuria. J. Clin. Microbiol. 3:42-46. 2. Alwall, N., and A. Lohi. 1973. Factors affecting the reliability of screening tests for bacteriuria. Acta Med. Scand. 193:499-503. 3. Bush, V. N., G. L. Picciolo, and E. W. Chappelle. 1975.

4.

5. 6.

7.

8.

9. 10.

11. 12.

13.

The effect of growth phase and medium on the use of the firefly adenosine triphosphate (ATP) assay for the quantitation of bacteria, p. 35-41. In E. W. Chappelle and G. L. Picciolo (ed.), Analytical applications of bioluminescence and chemiluminescence. NASA Scientific and Technical Information Office, SP-388, Washington, D.C. Conn, R. B., P. Charache, and E. W. Chapelle. 1975. Limits of applicability of the firefly luminescence ATP assay for detection of bacteria in clinical specimens. Am. J. Clin. Pathol. 63:493-501. Cowan, S. T., and K. J. Steel (ed.). 1%5. Manual for the identification of medical bacteria. Cambridge University Press, London. Curtis, G. D. W., and H. H. Johnston. 1979. A rapid screening test for bacteriuria, p. 448-457. In E. Schram and P. Stanley (ed.), Analytical applications of bioluminescence and chemiluminescence, Proceedings 1978. State Printing and Publishing, Inc., Westlake Village, Calif. Galen, P. S., and S. R. Gambino. 1975. Beyond normality. John Wiley & Sons, Inc., New York. Gutekunst, R. R. 1977. Clinical results using centrifugation and filtration procedures with the firefly luciferase ATP assay-detection of bacteria in urine and blood and antibiotic susceptibility testing, p. 491-524. In Proceedings from the Second Bi-annual ATP Methodology Symposium. SAI Technology Co., San Diego, Calif. Johnston, H. H., C. J. Mitchell, and G. D. W. Curtis. 1976. An automated test for the detection of significant bacteriuria. Lancet ii:400-402. Kass, E. H. 1957. Bacteriuria and the diagnosis of infections of the urinary tract. Arch. Intern. Med. 100:709-714. Nichols, W. W., G. D. W. Curtis, and H. H. Johnston. 1981. The identity and properties of firefly luciferase inhibitors in urine. Anal. Biochem. 114:443-440. Picciolo, G. L., E. W. Chappelle, E. A. Knust, S. A. Tuttle, and C. A. Curtis. 1975. Problem areas in the use of firefly luciferase assay for bacterial detection, p. 1-26. In E. W. Chappelle and G. L. Picciolo (ed.), Analytical applications of bioluminescence and chemiluminescence. NASA Scientific and Technical Information Office, SP388, Washington, D.C. Thore, A., S. Ansehn, A. Lundin, and S. Bergman. 1975. Detection of bacteriuria by luciferase assay of adenosine triphosphate. J. Clin. Microbiol. 1:1-8.


tic test is the predictive value of the test result. This value depends on the prevalence of the condition (e.g., bacteriuria) detected by the test. Depending on whether the test is going to be used for screening purposes, i.e., to sort out non-pathological specimens, or for diagnostic purposes, i.e., to positively identify a patient afflicted by that particular condition, demands on specificity and sensitivity differ. The luciferase test for bacteriuria has been suggested as a screening method. For this purpose, the demand on sensitivity is high and would correspond to the lower ATP limit of 13.5 nM or possibly even lower. If, on the other hand, the purpose of the test is to sort out patients with bacteriuria and to initiate treatment based on the test result, the demand on specificity is high and would correspond to the higher ATP limit of 67.5 nM or possibly even higher. Thus, the ultimate choice of criteria for a positive test will depend upon the purpose of the test and the prevalence in the clinical material at hand. It is suggested that further work regarding the luciferase bacteriuria test be aimed at designing a test suited for one of the outlined purposes and that cutoff limits and a practical design of the test be adapted to the chosen purpose.

J. CLIN. MICROBIOL.