Gonozyme

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Principles and methods
CRITICAL REVIEW
Anthony J. Barletta, M.D.
January, 1985
Gonozyme"
Gonozyme1" (Abbott Laboratories) is a solid-phase enzyme immunoassay (EIA) for
the rapid detection of gonococcal antigens in clinical specimens. In this system, a
specially treated polystyrene bead adsorbs gonococcal antigen present in the test
solution. The bead is washed to remove unbound material, incubated first with
rabbit antibody to Neisseria gonorrhoeae, and subsequently with goat anti-rabbit
serum antibody which is conjugated to horseradish peroxidase. The presence of this
enzyme on the bead surface is detected by a final incubation of the washed bead
with a peroxidase substrate (O-phenylenediamine containing hydrogen peroxide). If
the specimen is positive, a yellow-orange color reaction occurs. The intensity of the
color reaction is quantitated with a spectrophotometer (measured optical density at
492 nm) and is proportional to the quantity of gonococcal antigen adsorbed by the
bead. The mean absorbance of three negative controls plus 0.190 is the
recommended cutoff of positivity (2).
Summary of clinical evaluations
Several clinical evaluations of this assay have been published in the recent literature
(see references). These compare Gonozyme results with those of standard culture
techniques and/or direct microscopy of Gram-stained smears, utilizing the
percentage of positive cultures as an estimate of gonococcal disease prevalence.
The available data indicate that the Gonozyme1" assay is capable of rapidly
detecting gonococcal antigen in urethral and endocervical swab specimens as was
intended by the manufacturer, although some results indicate lower sensitivity,
specificity, and predictive values than were initially obtained (Table I). In general,
the reported sensitivity and specificity of EIA are comparable to those of the Gram
stain in males, whereas in females EIA appears more sensitive though less specific
than Gram stain (1,3,6,13,16,18,20).
Technical advantages and limitations
Numerous selective culture media and transport systems have been developed for
the isolation of gonococci. When a single swab specimen is collected, cultures can
optimally detect close to 95% of male urethral infections and 85% of female
cervical infections (4). Available culture techniques have several drawbacks
including: 1) loss of viability of gonococci under suboptimal transport or growth
conditions, 2) inability to grow the organisms after initiation of antibiotic therapy,
3) failure to culture vancomycin-sensitive strains or other strains with fastidious
growth characteristics, and 4) a time requirement of up to 2 to 3 days before results
are available (4).

Page 2
EIA systems are theoretically not affected by sensitivity of gonococci to
vancomycin or loss of viability in specimen transport. Organisms are rendered non
viable by the specimen preservative and dilution buffer included in the test kit.
This, however, precludes susceptibility testing and the identification of
penicillinase-producing gonococci if EIA is utilized as the sole diagnostic test.
Infections with small numbers of organisms may yield false negative results, and
there is at least a theoretical potential for false positive results in evaluating
patients for test of cure after therapy, due to detection of non-viable organisms.
Gonozyme has not yet been evaluated with pharyngeal or rectal specimens. Crossreacting
antigens from other Neisseria species and enteric organisms may pose
problems for its usefulness in evaluating specimens from these sites (3,21). Calcium
alginate or dacron swabs should not be used for collecting EIA specimens (2).
Does the Gonozyme EIA improve upon current diagnostic methods?
For males with urethritis, the Gram stain remains an inexpensive technique
providing rapid, accurate results when performed by experienced personnel (3,20).
In a hypothetical cost-benefit analysis, it seems difficult to justify substituting
Gonozyme for routine screening or first evaluation of symptomatic males. In
asymptomatic males the sensitivity of the Gram stain drops to 40-60% compared
with culture (8). In this group Gonozyme would be of greater potential value,
r especially when specimens require transport to a referral laboratory. Note however
that the test's sensitivity and positive predictive value in Stamm's series are lowest
(67 and 30% respectively) in men without urethral discharge in whom the prevalence
of disease was only 2% (20). This is in keeping with Bayes Theorem stating that
without perfect specificity, the predictive value of a positive test declines rapidly
with decreasing prevalence of disease in the test population.
Patients with asymptomatic gonorrhea presumably have fewer gonococci and hence
less antigen per swab. Loss of antigen in preparing Gram-stain smears and streaking
cultures plates prior to antigen assay may be a factor in reducing sensitivity of the
test in patients with few organisms to begin with. Along these lines, Schachter et
al. have demonstrated significant decreases in EIA sensitivity with multiple uses of
the same swab prior to inoculating the Gonozyme transport medium, and
recommends use of separate swabs for Gram-stains or cultural duplication (18).
In women with cervical gonorrhea diagnosis is confounded by the non-specific signs
and symptoms of infection and the relatively poor sensitivity of cervical Gram-stain
(approximately 42%) (3). A rapid diagnostic test more sensitive than the Gram-stain
would be of particular value in females who are at greatest risk of complication
from delayed therapy. In a recent study conducted at a sexually transmitted disease
clinic, 7.3% of women with gonococcal cervicitis not treated at the first visit
developed pelvic inflammatory disease before the followup visit after a mean
interval of 7 days (11).
Although more sensitive, EIA is not equivalent in speed or cost to the Gram stain —
^^ the assay takes approximately one hour and would likely be performed in batches
f^ once or twice daily. The reported sensitivity of Gonozyme in detecting cervical

n
Page 3
gonorrhea in some series seems unacceptably low for screening high risk populations
such as those encountered in sexually transmitted disease clinics. The available
data also indicate that the Gonozyme assay lacks sufficient positive predictive
value to replace culture as the definitive diagnostic test in female populations with
a low disease prevalence.
A positive Gonozyme assay is considered sufficient evidence of gonorrhea in males,
and is as accurate as a Gram-stain. As stated by the manufacturer, however,
positive Gonozyme results in females should be regarded only as presumptive and
confirmed by appropriate methods (2).
References
1. Aardoom HA, DeHoop D, Iserief COA, et al.: Detection of Neisseria
gonorrhoeae antigen by a solid-phase enzyme immunoassay. Br J Vener Dis
58:359-362, 1982.
2. Abbott Laboratories, North Chicago, Illinois: Gonozyme Diagnostic Kit:
Enzyme immunoassay for the detection of Neisseria gonorrhoeae in urogenital
swab specimens (package insert). September 1983.
3. Burns M, Rossi PH, Cox DW, et al.: A preliminary evaluation of the
Gonozyme test. Sex Transm Dis 10:180-183, 1983.
4. Carlson BL, Haley MS, Tisei NA, McCormack WM: Evaluation of four methods
for isolation of Neisseria gonorrhoeae. J Clin Microbiol 12:301-303, 1980.
5. Dans PE, Rothenberg R, Holmes KK: Gonococcal serology: how soon, how
useful, and how much? J Infect Dis 135:330-334, 1977.
6. Danielsson D, Moi H, Forslin L: Diagnosis of urogenital gonorrhea by
detecting gonococcal antigen with a solid phase enzyme immunoassay
(Gonozyme). J Clin Pathol 36:674-677, 1983.
7. Demetriou E, Sackett R, Welch DF, Kaplan DW: Evaluation of an enzyme
immunoassay for detection of Neisseria gonorrhoeae in an adolescent
population. JAMA 252:247-250, 1984.
8. Handsfield HH, Lipman JP, Harnisch E, et al.: Asymptomatic gonorrhea in
men. Diagnosis, natural course, prevalence and significance. N Engl J Med
290:117-123, 1974.
9. Judson FN: A clinic-based system for monitoring the quality of techniques for
the diagnosis of gonorrhea. Sex Transm Dis 5:141-145, 1978.
10. Judson FN, Maltz AB: A rational basis for the epidemiologic treatment of
gonorrhea in a clinic for sexually transmitted diseases. Sex Transm Dis
5:89-92, 1978.

Page 4
11. Lossick JG, Smeltzer MP, Curran JW: The value of the cervical Gram stain in
the diagnosis and treatment of gonorrhea in women in a venereal disease
clinic. Sex Transm Dis 9:124-127, 1982.
12. Martin R, Wentworth BB, Coopes S, Larson EH: Comparison of Transgrow and
Gonozyme for the detection of Neisseria gonorrhoeae in mailed specimens. J
Clin Microbiol 19:893-895, 1984.
13. Manis RD, Harris B, Geiseler PJ: Evaluation of Gonozyme, an enzyme
immunoassay for the rapid diagnosis of gonorrhea. J Clin Microbiol
20:742-746, 1984.
14. Mirrett S, Reller LB, Knapp JS: Neisseria gonorrhoeae strains inhibited by
vancomycin in selective media and correlation with auxotype. J Clin
Microbiol 14:94-99, 1981.
15. Nachamkin I, Sondheimer SJ, Barbagallo S, Barth S: Detection of Neisseria
gonorrhoeae in cervical swabs using the Gonozyme enzyme immunoassay.
Am J Clin Pathol 82:461-465, 1984.
16. Papasian CJ, Bartholomew WR, Amsterdam D: Validity of an enzyme
immunoassay for detection of Neisseria gonorrhoeae antigens. J Clin
Microbiol 19:347-350, 1984.
17. Rudrik JT, Waller JM, Britt EM: Efficacy of an enzyme immunoassay with
uncentrifuged first voided urine for detection of gonorrhea in males. J Clin
Microbiol 20:577-578, 1984.
18. Schachter J, McCormack WM, Smith RF, et al.: Enzyme immunoassay for
diagnosis of gonorrhea. J Clin Microbiol 19:57-59, 1984.
19. Sng EH, Rajan VS, Yeo K, Goh A: The recovery of Neisseria gonorrhoeae from
clinical specimens: effects of different temperatures, transport times, and
media. Sex Transm Dis 9:74-78, 1982.
20. Stamm WE, Cole B, Fennell C, et al.: Antigen detection for the diagnosis of
gonorrhea. J Clin Microbiol 19:399-403, 1984.
21. Tramont EC, Sandoff JC, Artenstein MS: Cross-reactivity of Neisseria
gonorrhoeae and the nature of the antigens involved in the bacteriocidal
reaction. J Infect Dis 130:240-246, 1974.

Table I. Clinical evaluations of Gonozyme1" EIA compared with culture in the detection of N. gonorrhoeae from urethral and endocervical swab specimens.
Author
Aardoom et al.
Abbott, Inc.
Hums et al.
Janielsson et al.
Demetriou et al.
ivlanis et al.
Nachamkin et al.
Papasian et al.
Rudriok et al.
Schachter et al.
Stainm et al.
Sex Sensitivity (%)
F ( p r o s t i t u t e s ) 6 2 - 1 0 0
F ( c o n t a c t s ) 6 0 - 9 8
M ( u r e t h r i t i s ) 9 0 - 1 0 0
F ( h i g h r i s k ) 8 7 . 2
F ( l o w r i s k ) 1 0 0
M ( h i g h r i s k ) 9 3 . 2
F 8 8 . 5
M 1 0 0
F 9 0 . 9
M 8 3
M (corrected for FP cultures) 85.7
F 8 7 . 5
M 1 0 0
F 9 6 . 4
M 9 7 . 1
F 8 7 . 2
Al
F 79.2
M 97.3
F 86.7
:V1 100
M (FVU) 91.6
F (Boston - 1 culture) 70
F (Boston - 2 cultures) 38.5
F (Richmond - I culture) 86.8
.vi (Boston + Richmond) 93.3
F 78
M (Total) 94
M (w/ urethral discharge) 95
M (w/o urethral discharge) 67
-1|
Specificity (%)
91-99
76-97
81-100
95.5
97.4
98.3
94.3
96.8
100
94.3
96.6
98
100
86.5
96.9
89.1
87.2
95.8
93.7
97.6
97.9
97.7
94.1
97.9
100
98
98
98
98
Positive
Predictive
Value (%)
78.6
76.5
100
82.5
25
96.8
90.5
97
100
66.7
80
85.7
100
62.2
94.3
37.2
71.3
96.5
(76.5)
(95.8)
(91.7)
94.4
71.4
97
100
85
90
97
30
Negative
Predictive
Value (%)
98.9
94.6
100
96.9
100
99.1
93.6
100
99.3
97.7
97.7
98.2
100
99.0
98.4
Disease
Prevalence
(% culture-positive)
11.8
27.7
67.3
16.96
0.85
34.8
-
7.3
11.9
Total
No. of
Patients
102
54
52
1,892
236
985
368
71
150
101
14.3 782
57
18.8
34.9
591
295
98.9 6.9 1,588
91.3
96.8
(93.7)
(100)
(98)
86
80
90
89.4
96
99
97
99
28.6 252
54.3 208
(19.2) 78
(35.9) 44
(20) 120
35.3 68
27.6 47
44.2 86
64.1 117
15 723
16
36
2
1,171
-
-

Table § Clinical evaluations of Gonozyme EIA compared with ^ture in the detection of N. gonorrhoeae from urethral |endocervical swab specimt |
Author
Aardoom et al.
Abbott, Inc.
Burns et al.
Danielsson et al.
Demetriou et al.
Manis et al.
Nachamkin et al.
Papasian et al.
Rudrick et al.
Schachter et al.
Stamm et al.
F (prostitutes)
F (contacts)
M (urethritis)
F (high risk)
F (low risk)
M (high risk)
F
M
S e x S e n s i t i v i t y ( % ) S p e c i fi c i t y ( % )
F MM (corrected for F P cultures)
F M
F
M
F M
F
M
F
M
M (FVU)
F (Boston - 1 culture)
F (Boston - 2 cultures)
F (Richmond - 1 culture)
M (Boston + Richmond)
F
M (Total)
M (w/ urethral discharge)
M (w/o urethral discharge)
62-100
60-98
90-100
87.2
100
98.2
88.5
100
90.9
83
85.7
87.5
100
96.4
97.1
87.2
79.2
97.3
86.7
100
91.6
70
38.5
86.8
93.3
78
94
95
67
91-99
76-97
81-100
95.5
97.4
98.3
94.3
96.8
100
94.3
96.6
98
100
86.5
96.9
89.1
PPositive o s i t i v e N e g a t i v e '
'
"
"
D
i s e
Negative
e
a s
s
e
Predictive Predictive ' (% culture-positive)
Prevalence
Value (%) Value (%) ;
78.6
76.5
100
82.5
25
96.8
90.5
97
100
66.7
80
85.7
100
62.2
94.3
37.2
98.9
94.6
100
96.9
100
99.1
93.6
100
99.3
97.7
97.7
98.2
100
99.0
98.4
11.8
27.7
67.3
16.96
0.85
34.8
-
7.3
11.9
Total
No. of
Patients
102
54
52
1,892
236
985
368
71
150
101
14.3 782
57
18.8
34.9
591
295
98.9 6.9 1,588
87.2 71.3 91.3 28.6 252
95.8 96.5 96.8 54.3 208
93.7 (76.5) (93.7) (19.2) 78
97.6 (95.8) (100) (35.9) 44
97.9 (91.7) (98) (20) 120
97.7 94.4 86 35.3 68
94.1 71.4 80 27.6 47
97.9 97 90 44.2 86
100 100 89.4 64.1 117
98 85 96 15 723
98 90 99 16 1,171
98 97 97 36 -
98 30 99 2 -