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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.

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 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.

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 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.

Endemic nosocomial Legionnaires disease has occurred at our medical center for several years. Two subtypes of Legionella pneumophila serogroup 1 (UH-1 and RH-1) have been isolated in approximately equal numbers from hospital potable water. However, almost all clinical isolates have been UH-1. To assess potential differences in virulence, 50% lethal doses (LD50) and 50% infective doses (ID50) of UH-1 and RH-1 were determined by intraperitoneal infection in guinea pigs. The UH-1 LD50 was 7.41 X 10(6) CFU, which was significantly lower than the RH-1 LD50 of 9.12 X 10(7) CFU (P = 0.0001). The mean time to death in UH-1-infected guinea pigs was also significantly shorter than in RH-1-infected animals (P = 0.0008). The UH-1 ID50 was 5.8 X 10(3) CFU, and although it was lower than the RH-1 ID50 of 1.4 X 10(4) CFU, this difference was not statistically significant (P = 0.21). This study demonstrates a difference in virulence between UH-1 and RH-1 in guinea pigs. Differences in strain virulence, as demonstrated between these two subtypes, may help to explain the widespread isolation of L. pneumophila from the environment contrasted with the limited occurrence of human Legionnaires disease.