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The Limulus amebocyte lysate (LAL) assay is currently being studied for use in diagnosing gonococcal urethritis/cervicitis syndromes and as a screening test for gonococcal cervicitis. Experience shows that the test can be adapted to office use by private physicians, providing rapid, accurate results in situations where laboratory facilities are unavailable. Further modifications are improving the speed and interpretation of results.

A test device incorporating Limulus amoebocyte lysate (Mallinckrodt, Inc., St. Louis, Mo.) was developed for the rapid, presumptive diagnosis of gonococcal and nongonococcal disease in males. The device, which was evaluated in 550 men with exudative urethritis, consisted of a specimen collection syringe, a dilution reservoir containing 10 ml of pyrogen-free water, and a Limulus amoebocyte lysate single-test vial. After specimen collection, the syringe was affixed to the dilution reservoir for rapid, accurate dilution of the clinical sample. Contamination of the specimen and potential biohazards to the user were prevented. The diluted sample was then transferred (via the collection syringe) to the lysate test vial for assay of endotoxin. Various incubation times at 37 degrees C were also studied in an additional 301 male patients, and time was reduced from the standard 60 to 30 min while still retaining equivalent predictability of culture results (P less than 0.05). Of the 550 males evaluated with the test device, 366 had positive cultures for Neisseria gonorrhoeae, and 184 were negative. A sensitivity of 99.2% and a specificity of 96.7% were obtained with the test device. Overall ability to predict culture results was 98.4%. Gram-stain sensitivity and specificity were 96.4% and 99.5%, respectively, with an overall accuracy of 97.5%. There were no statistical differences between the Limulus amoebocyte lysate test and Gram stain in predicting cultures (P less than 0.05). Thus, use of the Limulus amoebocyte lysate test device would enable the private physician to make an accurate, presumptive diagnosis of gonococcal and nongonococcal disease in males with exudative urethritis within 30 min without the need of a microscope and to initiate proper therapy during the patient's initial evaluation.

The Limulus amoebocyte lysate (LAL) assay was used to evaluate 115 females who were named as sexual contacts by men with culture-proven gonorrhea. These patients were treated for gonorrhea before laboratory confirmation, as recommended by the Centers for Disease Control, because of the lack of rapid screening tests and the serious consequences of undetected infection. For the LAL assay, endocervical samples were collected with depyrogenated cotton-tipped swabs, and the swabs were placed in 10 ml of diluent to assay for endotoxin; the negative predictive value of the LAL assay at this dilution was 100%. Incubation was carried out at 37 degrees C for 30 min; positive or negative results were indicated by gelation or lack of gelation, respectively. Lysate sensitivity was 0.3 ng/ml, with an Escherichia coli endotoxin standard. Single endocervical cultures and the LAL assay were both positive in 71 patients, but the Gram stain was positive in only 36 (50.7%) of these cases. For the 44 culture-negative cases, the LAL assay was negative in 21 (47.7%). Thus, the LAL assay was able to selectively exclude approximately half of the culture-negative gonorrhea contacts and would have spared these patients inappropriate therapy and contact tracing, without excluding culture-positive gonorrhea cases.

Cefaclor, a new orally administered cephalosporin, was evaluated by a randomized trial for effectiveness in the treatment of uncomplicated urethritis due to Neisseria gonorrhoeae in men. Regimens included 2,3, and 4 g of cefaclor, with or without 1 g of orally administered probenecid, as single daily doses for three days. The diagnoses were confirmed by isolation of N. gonorrhoeae; cures or therapeutic failures were determined by follow-up cultures on day 7 after completion of therapy. Sixty-six (73%) of 90 treated patients were evaluable for efficacy. The bacteriologic cure rate was 98% (65/66); one patient treated with 2 g of cefaclor plus probenecid had a positive culture for N. gonorrhoeae on follow-up examination. Adverse reactions consisted of mild nausea in five patients (7%) and vomiting in one patient (1%) who received 3- or 4-g doses. No treatment was discontinued, and no abnormality of screening hematologic tests or enzymes was observed. Thus, cefaclor, given in multiple doses, was highly efficacious for treatment of uncomplicated gonococcal urethritis in men.

Three Limulus amoebocyte lysate (LAL) preparations obtained from three different suppliers were comparatively evaluated for sensitivity to native endotoxin contained in urethral exudates from 28 men with gonococcal urethritis and 16 men with nongonococcal urethritis. One LAL preparation was not extracted with organic solvents during manufacture, whereas the other two were extracted with chloroform. All three LAL preparations had equivalent sensitivities (0.06 ng/ml) to an established reference endotoxin standard (EC-2), but significant differences in sensitivities were found among the different LAL preparations when testing clinical specimens. Dilution breakpoints of urethral samples for maximum sensitivity and specificity ranged from 1:400 to 1:1,600, depending on the LAL preparation. The nonextracted lysate was significantly more sensitive to the presence of endotoxin in gonococcal exudates than the other two preparations (P less than 0.001) but not significantly different from one LAL preparation (P greater than 0.05) in detecting endotoxin in nongonococcal exudates. An additional 116 men, 61 with culture-proven gonococcal urethritis and 55 with nongonococcal urethritis, were evaluated with three lots of nonextracted lysate with sensitivities ranging from 0.04 to 0.06 ng/ml, reference endotoxin EC-2. At a dilution breakpoint of 1:1,600, the sensitivity of the LAL test was 100%, and the specificity was 96%.

The limulus lysate assay was utilized as a diagnostic adjunct in the evaluation of three cases of disseminated gonorrhea in women. Although not a specific test for Neisseria gonorrhoeae, the limulus lysate assay, when used with properly diluted endocervical samples, gave results that correlated with conventional diagnostic techniques. If the advantages and limitations of the limulus lysate assay become fully appreciated, it may serve as a useful clinical tool for diagnosis of this syndrome.

A chromogenic substrate was used with Limulus amoebocyte lysate (LAL) and compared by parallel testing with the traditional gelation LAL method for the rapid evaluation of exudative urethritis in 125 male patients. Of these patients, 67 had positive cultures for Neisseria gonorrhoeae and 58 were negative. The corresponding prevalence of gonococcal urethritis was 53.6%. For assay, diluted urethral samples and chromogenic substrate were added directly to single-test LAL vials, and objective color endpoint determinations were made visually after a 10-min incubation period at 37 degrees C. Sensitivity and specificity were 98.5% and 93.1%, respectively, with an overall accuracy in predicting culture results of 96.0%. The predictive value of a positive LAL test was 94.3% in our patient population; in a population with a prevalence of gonococcal urethritis of only 10%, the predictive value would be 61.3%. Results were not statistically different from those obtained by the 30-min gelation LAL method or by Gram-stained smears read by experienced microscopists (P greater than 0.05). Unlike the delicate gel, the color endpoint was not prone to accidental mechanical disruption during incubation or reading. Thus, use of a chromogenic substrate greatly improved the utility and speed of the LAL assay for evaluating men with exudative urethritis while not affecting the accuracy of the test.

Eighty-one men with exudative urethritis were evaluated on initial visit for gonococcal urethritis by using the standard tube and newly developed microdilution Limulus amoebocyte lysate techniques. Serial dilutions of clinical specimens ranging from 1:100 to 1:102,400 were each tested, and results correlated with Gram stain and culture. Overall accuracy for predicting culture results was 98% for a dilution of 1:200 and 99% for a dilution of 1:400 for the tube and microdilution techniques, respectively. The microdilution technique predicted culture results in 98% of cases for dilutions ranging from 1:400 to 1:1,600, whereas the tube technique was as accurate for dilutions of only 1:100 and 1:200. The microdilution Limulus amoebocyte lysate technique was a rapid, reliable, sensitive, and economical diagnostic aid in the initial evaluation of exudative urethritis in men.

The Limulus amoebocyte lysate (LAL) assay was improved for the rapid evaluation of exudative urethritis in males. Two hundred men with various quantities of urethral discharge were evaluated. Pyrogen-free Dacron swabs were used for sample collection, and a chromogenic substrate was used for visible endpoint determination after a 10-min incubation. Of the 200 patients studied, 57 (29%) had minimal urethral discharges (less than 15 microliter) and could not be evaluated with Gonoscreen (Mallinckrodt, Inc., St. Louis, Mo.), an LAL test kit for the evaluation of urethritis which involves gentle aspiration for sample collection. The improved LAL assay had a sensitivity and specificity of 95 and 97%, respectively, and an overall accuracy to predict culture results of 96%. These results were not statistically different from Gram-stained smears read by experienced microscopists or from culture results (P greater than 0.05). Prevalence of gonorrhea in the study population was 40%, and the positive predictive value of the LAL assay was 95%. Thus, the use of swabs facilitated sample collection and increased by 29% the number of patients which could be evaluated with the LAL assay. In addition, the use of a chromogenic substrate reduced incubation time by 67% (30 to 10 min) and provided an objective color endpoint.

In an evaluation of the limulus assay as a method for detecting endotoxin in urethral exudates, positive results of urethral samples at a 1/200 dilution were obtained from 73 out of 73 patients with culture-positive gonococcal urethritis while negative results were obtained from 26 out of 27 patients with cuture-negative urethral specimens. A specimen from one patient, which gave negative results on Gram stain and culture, gave positive results to the limulus test. The overall accuracy of the limulus test for predicting culture results was 99% (p less than 0.001). Thus, in preliminary studies of otherwise healthy men, the results of the limulus assay correlated with those of biological methods for diagnosing urethral gonorrhoea; the test may, therefore, be of use in identifying cases of nongonococcal urethritis.

The Limulus amoebocyte lysate (LAL) assay was evaluated for its ability to detect or exclude gonococcal cervicitis in two groups of women. The first (positive) group consisted of 100 untreated women who were referred to the venereal disease clinic with culture-proven gonococcal cervicitis. The second (negative) group consisted of 50 normal volunteers who were evaluated on two separate occasions. In the first group, Gram stains and repeat cervical cultures were 53 and 93% sensitive, respectively. In the second group, Gram stains and cultures were negative. For the LAL assay, ectocervical mucus was removed with a sponge, and a depyrogenated cotton-tipped swab was then used to collect endocervical specimens. The swab was placed in 1 ml of diluent (1:1 dilution), and serial twofold dilutions were made and tested for endotoxin by the LAL assay. Incubation was carried out at 37 degrees C for 30 min; positive or negative results were indicated by gelation or lack of gelation, respectively. At a dilution of 1:256, sensitivity and specificity of the LAL assay were 57 and 99%, respectively. The positive predictive values ranged from 36.5 to 97.4% for theoretical prevalence rates of 1 to 40%. At a dilution of 1:8, the sensitivity and specificity were 100 and 78%, respectively. At this dilution, the negative predictive value was 100% regardless of the prevalence rate. Thus, these preliminary results show that at the higher dilution, the LAL assay was comparable to Gram stain in diagnostic accuracy of gonococcal cervicitis, and if used as a screening test at the lower dilution, a negative LAL assay would exclude women without gonococcal cervicitis.

In an evaluation of the limulus lysate assay (LLA) as a method for detecting gonococcal endotoxin in cervical exudates diluted 1:800, positive LLA results were obtained from 17 of 18 patients (94%) with culture-proved gonococcal cervicitis, and negative results were obtained from 22 of 22 patients (100%) with culture-negative specimens. In vitro tests comparing the sensitivity of the LLA for Neisseria gonorrhoeae and other gram-negative organisms showed the LLA to be more sensitive in detecting N. gonorrhoeae (minimum sensitivity, 10(4) organisms per milliliter) than other commonly encountered urogenital gram-negative bacteria (minimum sensitivity, greater than 10(5) organisms per milliliter). Thus, in preliminary studies involving otherwise healthy women, the LLA appeared to correlate with bacteriologic methods for diagnosing gonococcal cervicitis and may aid in identifying nongonococcal cervicitis. In addition, the LLA was easy to perform, with test results available within an hour after the patient's initial examination.