Download Comparative Evaluation of the Limulus Assay and the Direct Gram

Comparative Evaluation of the Limulus Assay
and the Direct Gram Stain for Detection
of Significant Bacteriuria
JAMES
H. JORGENSEN, PH.D.,
AND PAMELA
M. JONES, M.T. (ASCP)
Departments of Pathology and Microbiology, The University of Texas Health Science Center at
San Antonio and the Bexar County Hospital, San Antonio, Texas 78284
ABSTRACT
Jorgensen, James H., and Jones, Pamela M.: Comparative evaluation of the
Limulus assay and the direct Gram stain for detection of significant
bacteriuria. Am. J. Clin. Pathol. 63: 142-148, 1975. A double-blind study
comparing the Limulus in-vitro endotoxin assay with the direct Gram stain of
uncentrifuged urine for detection of significant bacteriuria was performed.
One-thousand seventy-seven urine specimens were examined by the two
methods and the results compared with results of quantitative urine
cultures. Two hundred three samples produced growth of >10 5 organisms
per ml. urine. The Limulus assay detected 86.2% of these specimens, and
98.8% of urines that contained >10 5 Gram-negative bacilli per ml. The
Gram stain procedure detected only 69.5% of urines containing >10 5
organisms per ml. and 74.5% of specimens with >10 5 Gram-negative
bacteria per ml. urine. T h e Limulus assay demonstrated both greater
sensitivity and greater specificity than the Gram stain procedure. Moreover,
the Limulus test is much less susceptible to errors of interpretation than
methods involving microscopy. (Key words: Endotoxin in urine; Detection
of bacteriuria; Gram stain for bacteriuria; Limulus assay for bacteriuria.)
I N GENERAL, there have been three separate approaches to development of new
screening tests for the detection of significant bacteriuria. One approach has
been the introduction of miniaturized
inexpensive culture devices. 2,5 However,
these devices still require a traditional
culture incubation period of 18-24 hours.
A second approach has been the detection
of bacterial products or enzymes in urine
by chemical tests such as the Griess nitrate
Received J u n e 13, 1974, revised July 24, 1974;
accepted July 24, 1974.
Supported in part by NIH-General Research
Support Grant # 5 S01RR05654-05.
Address reprint requests to Dr. Jorgensen.
142
reductase test17 or the measurement of
tetrazolium reductase. 12 These tests have
the advantage of providing more rapid
results, but are often much less reliable. 6 ' 18
A third approach has been the visualization of bacteria in urinary sediment by
use of either the phase-contrast microscope 1 or a Gram stain of fresh uncentrifuged urine. 13 In particular, the Gram
stain has been reported to correlate with
significant colony counts in 7 5 - 9 5 % of
cases.3'11,12 However, considerable judgment must often be applied to the estimation of the number of organisms visualized in Gram-stained smears.
January 1975
143
DETECTION OF BACTERIURIA
The Limulus in-vitro assay is presently
the most sensitive method available for
the detection of endotoxin, 15 and can be
applied easily to the sampling of a variety
of biologic fluids.4,18 A previous study of a
limited number of selected patients indicated that the Limulus test could be
applied to the examination of urine for
the detection of endotoxin associated with
Gram-negative bacteria. 9 Based on this
preliminary study, the Limulus test appeared to be a promising screening test
for significant bacteriuria. The purpose of
the present investigation was to compare,
by use of a double-blind study, the
Limulus assay and the direct Gram stain
for rapid detection of significant bacteriuria in an expanded, unselected patient group.
Table 1. Criteria for Evaluation of Gram
Stains of Urinary Sediment
WBC's/Field
Classification
Classification scheme for leukocytes seen with 43 x
objective
>20
12-19
8-11
4-7
<4
4+
3+
2+
1+
Neg.
Cells/Field
Classification
Classification scheme for bacteria seen with 97 x oil
immersion objective
>10
6-9
2-5
1
<1
4+
3+
2+
1+
Neg.
Materials and Methods
Patients Surveyed
The patients in this study were those
individuals whose urine specimens were
submitted to the Microbial Pathology
Laboratory of the Bexar County Hospital
for routine quantitative bacteriologic culture. They included hospitalized patients
and patients seen in one of more than 50
outpatient clinics. In most cases, cleanvoided midstream urine was collected; in
certain other cases catheterized specimens
were obtained. Every effort was made to
process urine specimens within one hour
of collection, or they were refrigerated to
assure the accuracy of quantitation of
bacteria in the sample.
Cultures were incubated at 37 C. and
examined at 24 and 48 hours.
Performance of Limulus Assay
Limulus lysate was prepared according
to methods previously described. 10 Lysate
batches used in this study could detect as
little as 5 ng. per ml. of Escherichia coli
055:B5 endotoxin (Boivin extract, Difco
Laboratories). Prior to performance of
the Limulus assay, urine samples were
diluted 1:100 in pyrogen-free saline solution (Travenol Laboratories). A 0.1-ml
volume of the diluted urine was added to
a 0.1-ml. volume of Limulus lysate in
pyrogen-free disposable glass test tubes
(Corning Glass Works). The reaction mixture was then incubated for 2 hours at
37 C. A Limulus assay was considered
Culture Methods
positive only if a 3 + or 4 + reaction was obUrine specimens were cultured by use tained following the incubation period. 10
of a volume-calibrated platinum bac- Therefore, based on the lysate sensitivteriologic loop calibrated to deliver 0.001 ity (5 ng. per ml.) and the urine dilution
ml. of urine. 8 Samples were streaked onto factor (1:100), an equivalent endotoxin
one plate each of 7.5% sheep blood agar level of 500 ng. per ml. or more was
(Hyland) and MacConkey agar (Hyland). considered "positive" in this study.
144
A.J.C.P.—Vol.
JORGENSEN AND JONES
Table 2. Comparison of Limulus Assay
and Gram Stain Results on Urine
Specimens Containing >10 5
Organisms per ml.
Organism
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus mirabilis
Enterobacler aerogenes
Enterobacter cloacae
Serralia tnarcescens
Providencia spp.
Herellea vaginocola
Citrobacter freundii
Staphylococcus aureus
Group 1) Streptococcus
Group B Streptococcus
Streptococcus viridaus
Candida albicans
2 GNR'st
3 or >GNR's
2 CP'st
8 or >CP's
1 GNR + 1 GP
Mull. GNR's + GP's
TOTAL
Total
No.
Limulus
Positive*
96
17
8
6
2
2
1
1
1
1
3
2
1
3
3
21
6
5
•1
12
7
94
17
8
6
2
2
1
1
1
1
0
0
0
0
0
21
6
0
2
8
5
(97.9%)
(100.0%)
(100,0%)
(100.0%)
(100.0%)
(100,0%)
(100.0%)
(100.0%)
(100.0%)
(100.0%)
203
175
Gram Stain
Positive*
(100.0%)
(50.0%)
(61.5%)
(71.4%)
78
14
7
5
0
1
0
0
0
0
3
0
0
1
3
12
3
3
1
6
4
(86.2%)
141
(69.5%)
(100.0%)
(100.0%)
(81.3%)
(82.4%)
(87.5%)
(83.3%)
(50.0%)
(33.3%)
(100.0%)
(61.1%)
(50.0%)
(60.0%)
(25.0%)
(46.2%)
(57.1%)
* Percentages indicate <7c of totalnumber positive.
t GNR = Gram-negative rod: GP = Gram-positive organism.
Gram Stain of Urinary Sediment
Gram stains were prepared in the usual
manner following transfer of a 4-mm.
bacteriologic loopful of uncentrifuged
urine to a glass slide and allowing it to air
dry. Smears were examined with the
high-power objective (43 x) for the presence of leukocytes, followed by examination for bacteria using the oil-immersion
lens (97 X). The results of examination of
ten successive fields were recorded based
on the criteria outlined in Table 1. Smears
were considered "positive" if they could
be classified as 1 + or greater.
Definition of Significant Bacteriuria
T h e criterion for "significant bacteriuria" used in this study was a colony
count of >10 5 organisms per ml. urine, as
defined by Kass."
63
Table 3. Comparison of Limulus Assay
and Gram Stain Results on Urine
Specimens Containing 104—105
Organisms per ml.
Organism
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus spp.
Misc. GNR'st
Group D Streptococcus
Staphylococcus epidermidis
Group B Streptococcus
Streptococcus viridans
Candida albicans
2 GNR's
3 or >GNR's
2 GP'st
3 or >GP's
1GNR+1GP
Mult. GNR's + GP's
TOTAL
]Limulus
Positive*
Total
No.
16
3
3
3
5
10
7
2
3
2
7
2
18
5
13
12
111
7
2
1
2
0
0
0
0
0
0
2
1
5
1
2
3
Gram Stain
Positive*
(43.8%)
(66.7%)
(33.3%)
(66.7%)
2
1
0
0
0
0
0
0
0
0
1
0
4
1
1
2
(28.6%)
(50.0%)
(27.8%)
(20.0%)
(15.4%)
(25.0%)
26 (23.4%)
(12.5%)
(33.3%)
(14.3%)
(22.2%)
(20.0%)
(7.7%)
(16.7%)
12 (10.8%)
* Percentages indicate % of total number positive.
t GNR = Grain-negative rod: GP = Gram-positive organism.
Table 4. Correlation of Gram Stain and
Culture Results (>10 5 Organisms
per ml.)*
Organisms
Correct
Incorrect
Partly
Correct
GNR's
GPC's
GPR's
GN + GP
Yeast
110
3
0
0
3
0
6
3
0
0
5
4
1
5
1
* GNR = Gram-negative rod; GPC = Gram-positive coccus; GPR
= Gram-positive rod.
Controlling Bias
A double-blind protocol was employed
to minimize bias in comparing the results
of Limulus assay and the Gram stain with
results of quantitative cultures. The urine
cultures were performed in the usual
fashion by the routine clinical laboratory
technologists. Results of Limulus tests and
Gram stains were read and recorded
separately by the two authors prior to the
availability of the culture results.
January 1975
DETECTION OF BACTERIUR1A
Calculation of Sensitivity and Specificity
The definitions of sensitivity and specificity are those used by the World Health
Organization 19 : sensitivity = diseased persons with positive test/all persons in population with disease; specificity = non diseased persons with negative test/all persons in population without disease.
Results
One thousand seventy-seven (1,077)
urine specimens were examined. Two
hundred three (203) of the specimens
contained more than 100,000 organisms
per ml. urine, while 111 contained between 10,000 and 100,000 per ml. Totals
of 229 positive Limulus assays and 209
positive Gram stains were obtained.
One hundred forty-six (146) of 203
specimens containing >10 5 organisms per
ml. produced grwoth of pure cultures of
the organisms shown in Table 2, with
Escherichia coli being the most frequent
isolate. One hundred and thirty-two (132)
of these specimens had positive Limulus
tests (132/146 or 90.4%) and 112 had
positive Gram stains (112/146 or 76.7%).
Fifty-six (56) of the 203 urines containing
>10 5 organisms per ml. comprised mixed
cultures of two or more organisms. Mixed
cultures of Gram-negative rods totaling
>10 5 per ml. were detected in all cases by
the Limulus assay, while only 15 of 27
were detected by the Gram stain. However, more false-negative results were
obtained with the Limulus test than with
the Gram stain procedure with mixed
cultures comprised chiefly of Grampositive organisms.
Table 3 describes results of Limulus
assays and Gram stains of urine specimens
containing 104— 10s organisms per ml. Of
the 111 specimens, 26 (23.4%) yielded
positive Limulus tests, while only 12
specimens (10.8%) were recorded as positive using the direct Gram stain.
145
Table 5. Correlation of Pyuria* with
Bacteriuriaf
WBC's
Culture
>10 5
per ml.
Culture
10 4 -10 5
per ml.
Culture
<10 4
per ml.
4+
3+
2+
1+
4
5
15
28
1
1
4
4
3
3
2
5
Total positive
Negative
52
151
10
101
13
750
* Pyuria defined as 4 or more WBC's per high-power field of
uncentrifuged Grani-slrained urine.
t Bacteriuria defined as > 100.000 organisms per nil. urine.
T h e correlation between culture results
and organisms visualized on positive
Gram stains is outlined in Table 4. Results
of Gram stains and cultures were reviewed to determine whether a specimen
that grew >10 5 E. coli had a corresponding Gram stain report indicating that
Gram-negative rods were seen, as opposed to diphtheroid-like organisms or
Gram-positive cocci, for example. Therefore, Gram stain results were graded
either correct, incorrect, or partly correct
(in the case of some mixed cultures). It
should be noted that proportionately
more incorrect responses were recorded
with Gram-positive cocci and rods than
with Gram-negative bacilli.
An attempt was also made to correlate
the presence of pyuria, as determined in
this study, with significant bacteriuria
(Table 5). Twenty-five (25) per cent (52/
203) of specimens with >10 5 organisms per
ml. had significant numbers of leukocytes
visualized on the Gram-stained smear.
Only 9% (10/111) of specimens containing
104— 10s organisms per ml. had significant
numbers of leukocytes. Less than 2%
(13/763) of specimens with <10 4 per ml.
had pyuria.
The overall correlation of quantitative
urine cultures with Limulus assays and
146
JORGENSEN AND JONES
Table 6. Overall Correlation of Urine
Cultures with Limulus Assays
and Gram Stains
Agreement with Culture
Limulus Assay
Gram Stain
s
Samples with > 10
organisms per ml.
86.2%
(175/203)
69.5%
(141/203)
Samples with > 10s
GNR'sperml.*
98.8%
(159/161)
74.5%
(120/161)
94.4%
(1,016/1.077)
88.5%
(953/1,077)
Overall ability to
classify urine specimens
correctly as < 10s per ml.
or>IO'perml.
* GNR = Gram-negative rod.
Gram stains is summarized in Table 6.
The Limulus assay detected 86.2% of
urine specimens containing > 1 0 5 organisms per ml., while the Gram stain
detected only 69.5%. In the case of urines
containing >10 5 Gram-negative bacilli per
ml., the Limulus assay detected 98.8%,
while 74.5% were detected by the Gram
stain. The overall ability to classify urine
specimens correctly as containing either
<10 5 or >10 5 per ml. indicates that the
Limulus assay correctly classified 94.4% of
all urines, whereas the Gram stain
method accurately classified 88.5% of the
samples.
Table 7 indicates the results of calculations of the sensitivity and specificity of
each test. The Limulus assay has a sensitivity of 86.2% and a specificity of
96.1%. T h e Gram stain procedure has a
sensitivity of 69.5% and a specificity of
92.8%.
Discussion
The need for periodic screening of
certaia high-risk groups such as pregnant
women, school girls, and diabetics for the
presence of significant bacteriuria has
been well established. 7,16 T h e pour-plate
quantitative culture has been accepted as
A.J.C.P.—Vol.
63
the most accurate laboratory procedure
available for documenting bacteriuria. A
streak plate made with a calibrated
platinum loop is a suitable alternate
method requiring less time and equipment. 7 However, both methods still require skill, time, and equipment not
readily available in all office or clinic
settings. Consequently, several miniaturized culture devices and chemical
tests have been proposed as practical
screening devices for bacteriuria. However, most of these procedures have met
with rather limited acceptance. 6
T h e findings of the present investigation confirm our previous preliminary
observations 9 that the Limulus in-vitro
endotoxin assay can be used successfully
for the detection of significant bacteriuria.
The results of the Limulus test can be
available within l-2'/2 hr. of collection of
a urine specimen. Although results of
Gram stains can be obtained in a shorter
period, more time is required of technical
personnel for preparation and interpretation of a Gram-stained smear than is
required for performance of a Limulus
assay. Furthermore, performance of the
Limulus test does not require the degree
of subjective judgment for accurate quantitation of bacteria required by methods
involving microscopy of urinary sediment.
This is emphasized by the considerably
greater numbers of both false-positive
and false-negative results observed with
the Gram stain.
The sensitivity and specificity of the
Limulus assay for endotoxin results in
detection of nearly 100% of urines containing >10 5 Gram-negative bacteria per
ml. A major deficiency of the Limulus
assay for this purpose is that Grampositive bacteria and yeast are undetectable, due to their lack of endotoxin. Therefore, the predictive value of the Limulus
assay for significant bacteriuria lies in the
normal preponderance of Gram-negative
uropathogens, which possess endotoxin.
January 1975
DETECTION OF BACTERIURIA
147
Table 7. Sensitivity* and Specificity*
The direct Gram stain of uncentrifuged
urinary sediment appears to have a
of the Limulus Assay and the
false-negative rate of approximately 30%
Direct Gram Stain
irrespective of the group of organisms
Limulus Assay
Gram Stain
involved. The data in Table 4 indicate
that actually more incorrect responses Sensitivity
86.2%
69.5%
96.1%
92.8%
regarding the basic identities of organisms Specificity
False-positive rate
3.9%
7.2%
seen in urinary sediment occurred with False-negative rate
13.8%
30.5%
Gram-positive organisms. Therefore, both
* Based on calculations described in text.
the Limulus assay and the direct Gram
stain procedure seem better able to define
bacteriuria accurately when Gram-nega- were further investigated by review of the
tive organisms are involved.
medical charts of the 27 patients comprisNeither the Limulus assay nor direct ing these two groups. Current antimicromicroscopy could be considered an ac- bial chemotherapy was a probable explaceptable method for detection of bac- nation for negative urine cultures from
teriurias involving 104—105 organisms per five of these patients. An additional eight
ml. However, the Limulus assay again patients with positive Limulus assays were
defined significantly more urines in this diabetic and might be expected to have an
group than was possible using the Gram increased incidence of bacteriuria; howstain.
ever, urine cultures failed to confirm this
The percentage agreement of the Gram supposition. No apparent explanation was
stain procedure with quantitative cultures found for the positive Limulus tests of
reported here is lower than that of certain urine from the remaining 14 patients.
previous studies. 3,8 However, in the study
Another important factor in the evaluaby Hoeprich, 8 a Gram stain was consid- tion of any potential screening test is the
ered predictive of bacteriuria of 10,000 cost of performing the procedure. The
organisms per ml. or more (and therefore cost of a Limulus assay as performed in
positive) "if bacteria were seen in nearly this study would be approximately 75^!
every field." Even with these less stringent per specimen. While this seems reasonacriteria, Hoeprich experienced a false- bly inexpensive, it cannot compare to the
positive rate of 11.5%. Brupacher and almost negligible cost of preparing a
associates 3 considered a Gram-stained Gram-stained smear.
smear positive if "any bacteria were
T h e lack of predictive value for bacnoted." The latter authors also considered teriuria obtained by visualization of
a colony count of 10,000 bacteria per ml. leukocytes in unsedimented urine is in
to be significant. Therefore, the more agreement with findings of the previous
stringent criteria for positivity (at least study of Pryles and Eliot.14 T h e data in
one organism per oil-immersion field and Table 5 emphasize that bacteriuria may be
at least 100,000 organisms per ml.) used present in the absence of pyuria as
in the present investigation probably ex- defined, and pyuria may be present withplain the lower predictive value of the out bacteriuria. Therefore, the visualizaGram stain in our hands.
tion of significant numbers of leukocytes
Specimens in this study which produced in urinary sediment may be suggestive of
growth of <10 4 organisms per ml. but urinary tract infection, but pyuria alone
which had either a positive Limulus test cannot be considered indicative of infecand a positive Gram stain or a positive tion.
Limulus test and a negative Gram stain
In conclusion, both the Limulus assay
148
JORGENSEN AND JONES
and examination of a Gram-stained smear
of urinary sediment can be used for
detection of significant bacteriuria with
reasonable success. The Limulus assay
shows both greater sensitivity and greater
specificity than the Gram stain procedure.
Furthermore, the Limulus assay seems
considerably less susceptible to subjective
errors of interpretation than methods
involving microscopy. Since the presence
of antimicrobial agents in urine does not
affect either the Limulus test or the Gram
stain, either test could be used to assess
the effectiveness of treatment of previously diagnosed urinary tract infections.
A disadvantage of both methods is that
with few exceptions, information regarding the identity of organisms or the
presence of mixed infections is not obtained.
Acknowledgments. T h e technologists and technicians
of the Microbial Pathology Laboratory of the Bexar
County Hospital assisted in the performance of this
investigation.
References
1. Brody LM, Webster C, Kark RM: Identification
of elements of urinary sediment with phase
contrast microscopy. JAMA 206:1777-1781,
1968
2. Brupacher R, Domingue G: Experiences with a
screening test for bacteriuria. Am J Clin
Pathol 59:203-210, 1973
3. Bulger RJ, Kirby MM: Simple tests for significant bacteriuria. Arch Intern Med 112:
156-160, 1963
4. Cooper JF, Hochstein HD, Seligmann EB Jr:
T h e Limulus test for endotoxin (pyrogen) in
radiopharmaceuticals and biologicals. Bull Parenter Drug Assoc 26:153-162, 1972
A J. C.P.—Vol. 63
5. Craig WA, Kunin CM: Quantitative urine culture method using a plastic "paddle" containing dual media. Appl Microbiol 23:919-922,
1972
6. Craig WA, Kunin CM, De Groot J: Evaluation
of new urinary tract infection screening devices. Appl Microbiol 26:196-201, 1973
7. Greenberg ND, Stamler J, Zackler J, et al:
Detection of urinary tract infections in pregnant women. Public Health Rep 8 0 : 8 0 5 811, 1965
8. Hoeprich PD: Culture of the urine. J Lab Clin
Med 56:899-907, 1960
9. Jorgensen J H , Carvajal HF, Chipps BE, et al:
Rapid detection of gram negative bacteriuria
by use of the Limulus endotoxin assay. Appl
Microbiol 26:38-42, 1973
10. J o r g e n s e n J H , Smith RF: Preparation, sensitivity
and specificity of Limulus lysate for endotoxin
assay. Appl Microbiol 26:43-48, 1973
11. Kass EH: Asymptomatic infections of the urinary tract. T r a n s Assoc Am Physicians
69:56-64, 1956
12. Neter E: Evaluation of the tetrazolium test for
the diagnosis of significant bacteriuria. JAMA
192:769, 1965
13. Pryles CV: T h e diagnosis of urinary tract infection. Pediatrics 26:441-451, 1960
14. Pryles CV, Eliot CR: Pyuria and bacteriuria in
infants and children. Am J Dis Child
110:628-635, 1965
15. Rojas-Corona R, Skarnes R, Tamakuma S, et al:
T h e Limulus coagulation test for endotoxin: A
comparison with other assay methods. Proc
Soc Exp Biol Med 132:599-601, 1969
16. Smith LG, Schmidt J: Evaluation of three
screening tests for patients with significant
bacteriuria. JAMA 181:431-433, 1962
17. Smith LG, Thayer WR, Malta EM, et al: Relationship of the Griess nitrite test to bacterial
culture in the diagnosis of urinary tract
infection. Ann Intern Med 54:66-72, 1961
18. Trippodo NC, Jorgensen J H , Priano LL, et al:
Cerebrospinal fluid levels of endotoxin during
entotoxemia. Proc Soc Exp Biol Med 143:
932-937, 1973
19. Wilson J M G . J u n g n e r G: Principles and Practice
of Screening for Disease. World Health Organization, Public Health Papers, Geneva,
1968