Comparative evaluation of the tube and microdilution Limulus lysate techniques for rapid presumptive diagnosis of gonococcal urethritis in men.

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.     

Response of several Limulus amoebocyte lysates to native endotoxin present in gonococcal and nongonococcal urethral exudates from human males.

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%.     

Rapid evaluation of gonococcal and nongonococcal urethritis in men with Limulus amoebocyte lysate and a chromogenic substrate

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.     

Lack of utility of a Limulus amoebocyte lysate assay in the diagnosis of urethral discharges in men.

We evaluated a Limulus amoebocyte lysate assay (LALA) test kit for the diagnosis of gonorrhea in 883 unselected men with urethral discharge. Results were compared with those of Gram-stained smears and Martin-Lewis cultures. Of 331 men with gonococcal urethritis and 552 men with nongonoccal urethritis, 125 (37.8%) and 503 (91.1%), respectively, could not be evaluated by LALA owing either to insufficient discharge specimen to perform the test (569 or 64.4%) or to other exclusion criteria (59 or 6.7%). Of 255 LALA-evaluable discharges, LALA correctly diagnosed 252 (98.8%), compared with 244 (95.7%) for the Gram-stained smear. However, the Gram-stained smear also correctly diagnosed 96.5% of 456 men with insufficient discharge for LALA testing. The clinical utility of the LALA test kit is severely limited by performance criteria that exclude the majority of unselected men with urethritis. In addition, it is more technically cumbersome, time consuming, and costly than Gram-stained smears. Further test modifications are unlikely to overcome these inherent disadvantages of LALA.     

Improved utility of Gonoscreen, a Limulus amoebocyte lysate assay, in the evaluation of urethral discharges 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.     

Chromogenic Limulus amoebocyte lysate assay for rapid detection of gram-negative bacteriuria.

A chromogenic Limulus amoebocyte lysate assay was evaluated as a rapid screening test for the detection of clinically significant gram-negative bacteriuria. The development of a distinctive yellow color after the addition of chromogenic substrate to the Limulus amoebocyte lysate-urine reaction mixture was used to measure greater than or equal to 10(5) gram-negative bacteria per ml. A total of 324 urine specimens were assayed, with 68 gram-negative urinary tract infections identified as defined by quantitative urine colony counts of greater than or equal to 10(5) bacteria per ml. Of these, 68 and 67 of 68 were detected by the chromogenic Limulus amoebocyte lysate assay at urine dilutions of 1:10 and 1:20, respectively. Nine false-positive chromogenic Limulus amoebocyte lysate assay results were observed at both urine dilutions and in the same specimens. At a urine dilution of 1:10, sensitivity and specificity were 100 and 96.6%, respectively, with predictive values of 100% for a negative test and 88.3% for a positive test. At a urine dilution of 1:20, sensitivity and specificity were 98.6 and 96.6%, respectively; predictive values were 99.6% for a negative test and 88.3% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for the diagnosis of clinically significant gram-negative bacteriuria.     

Comparison of plasma extraction techniques in preparation of samples for endotoxin testing by the Limulus amoebocyte lysate test.

Due to the presence of inhibitory and possible mimicking substances in plasma difficulties have occurred in the use of the Limulus amoebocyte lysate test. Currently, there are a variety of extraction techniques discussed in the literature which are used to remove these interfering substances, but there is little information comparing these techniques. Five such procedures were compared in their ability to provide an extracted plasma sample in which low levels of endotoxin could be detected by the Limulus amoebocyte lysate test. Results indicated that some procedures adversely affected endotoxin detection. The dilution + heating extraction method was found to be as effective as the widely used chloroform extraction method. Comparison of Limulus amoebocyte lysate test results from healthy human plasma samples extracted by these two methods indicated that lysate type and not extraction procedure was associated with previously reported questionable positive tests. Thus, ambiguities associated with Limulus amoebocyte lysate tests of plasma samples may be due not only to extraction method but also the lysate type employed.     

Comparison of the histamine hypersensitivity and the Limulus amoebocyte lysate tests for endotoxin activity.

The histamine hypersensitivity test and the Limulus amoebocyte lysate test were compared for their effectiveness to quantitate endotoxin activity. The two tests compared favorably in all the trials, except with a sample of endotoxin from Brucella abortus that gave a positive Limulus amoebocyte lysate test at a concentration of 0.001 microgram, while failing to sensitize mice to histamine at a dose of 16 microgram per mouse. The Limulus amoebocyte lysate test was more sensitive than the histamine hypersensitivity test.     

Positive Limulus amoebocyte lysate reactions with polyriboinosinic acid x polyribocytidylic acid.

The annealed copolymer polyriboinosinic acid x polyriboinosinic acid reacted with Limulus amoebocyte lysate to cause gelation at a concentration approximately 2,000-fold or greater than bacterial endotoxins. This copolymer was pyrogenic in rabbits and demonstrated hypochromicity, but no significant correlation was noted among Limulus amoebocyte lysate reactivity, progenicity, and hypochromicity. Like endotoxin, polyriboinosinic acid x polyribocytidylic acid did not react with purified Limulus coagulogen. Similar concentrations of the homopolymers polyriboinosinic acid and polyribocytidylic acid were negative or significantly below the Limulus amoebocyte lysate reactivity of the copolymear and essentially nonpyrogenic. Thus, polyriboinosinic acid x polyribocytidylic acid is a compound in addition to endotoxin that effects a positive Limulus amoebocyte lysate test.     

Clinical value of endotoxin determination in infection. Comparison of the Limulus amebocyte lysate test with detection of bacterial pathogens

To evaluate usefulness of Limulus amoebocyte lysate test and blood culture in the diagnosis of septicemia both tests were performed in 27 intensive care patients. Test results were compared with a clinical sepsis score. Ten (62%) out of 16 patients with clinical diagnosis of septicemia showed a positive endotoxin test and 11 (69%) a positive blood culture. In 14 patients (87%) either endotoxin test or blood culture revealed a positive result. Two out of 11 patients (20%) classified by the sepsis score as non-septic showed positive blood cultures as well as positive endotoxin tests. 4 patients with gram-positive bacteria in the blood cultures showed a positive endotoxin test. Due to lack of sensitivity and specificity the Limulus amoebocyte lysate test is of rather low value in the diagnosis of septicemia. Simultaneous performance of Limulus amoebocyte lysate test and blood culture is able to improve the sensitivity, which then over-rules the one obtained when only blood cultures are performed.     

Sensitivity, specificity, and predictive values of the Limulus lysate assay for detection of exclusion of gonococcal cervicitis

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.     

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.     

Survey for positive Limulus amoebocyte lysate test in plasma from humans and common research animals.

A survey for positive Limulus amoebocyte lysate tests was conducted on apparently healthy humans, mongrel dogs, rats, mice, rabbits, and squirrel monkeys. Only mongrel dog (45.8%) and human (32.8%) plasma samples gave positive tests. In dogs, a significant correlation between positive Limulus amoebocyte lysate tests and the presence of intestinal parasites was found. Positives found in human plasma samples were thought to be due to the presence of background levels of endotoxin or some possible mimicker substance found in the plasma after chloroform extraction. It was concluded that there was a need to distinguish between these positive Limulus tests and those which represent significant endotoxemia.     

Rapid detection of gram-negative bacterial peritonitis by the Limulus amoebocyte lysate assay.

The chromogenic Limulus amoebocyte lysate test effectively detected 66 (100%) culture-proven gram-negative peritonitis cases among 185 continuous ambulatory peritoneal dialysis patients with clinical evidence of infectious peritonitis.     

Augmentation of endotoxin fever by recombinant human beta interferon in rabbits.

Nonpyrogenic amounts of endotoxin (0.1 to 1 ng/kg), hardly detectable by conventional Limulus amoebocyte lysate tests, could produce a fever of around 1 degree C when injected with a nonpyrogenic dose (6 X 10(5) U/kg) of recombinant human beta interferon (IFN-beta) in rabbits. Release of endogenous IFN and tumor necrosis factor by endotoxin was also dramatically increased by recombinant human IFN-beta, and their levels in the blood were closely correlated with the increase of body temperature. These data suggest, if the synergism between IFN and endotoxin also operates in the homologous system (human IFN-human cells), that contaminating endotoxin in IFNs, even if not detectable by Limulus amoebocyte lysate test, can contribute to IFN fever to a considerable extent in humans.     

Co-existence of N. gonorrhoeae and U. urealyticum in male urethra

Eight hundred and forty male patients attending sexually transmitted disease (STD) clinic for urethritis were investigated. Out of them, 31.6% had gonococcal urethritis, 16.1% suffered from nongonococcal urethritis due to Ureaplasma urealyticum and in 12.6%, both the organisms were present. Though 14.62% strains of N. Gonorrhoeae were resistant to penicillin, all the strains were sensitive to spectinomycin; while all Ureaplasma strains were sensitive to tetracyclines. As the treatment differs for these two organisms, it is necessary to identify the correct etiological agent.     

Interactions of lipid a and liposome-associated lipid A with Limulus polyphemus amoebocytes.

Lipid A or lipid A fractions and liposomes containing lipid A were tested for the ability to gel Limulus amoebocyte lysates and for effects on intact Limulus amoebocytes. Liposomes having a relatively low concentration of lipid A did not produce coagulation of lysate and were designated as Limulus-negative, but liposomes having a high concentration of lipid A were Limulus-positive. Limulus-negative liposomes had no effect on intact amoebocytes. Limulus-positive liposomes caused a striking transformation in the appearance of amoebocytes in that the cells sent out long filamentous extensions that formed a tangled network of processes between cells. The filamentous projections were similar to those that have been previously observed in the presence of gram-negative bacteria. We conclude that amoebocytes have the ability to recognize Limulus-positive liposomes, but the lack of activation of Limulus lysate or the absence of amoebocyte recognition does not prove the absence of liposomal lipid A. We also found that individual lipid A fractions were heterogeneous in their ability to gel lysate. Of eight fractions tested, one (fraction 1) had no detectable activity above the background, and the seven others had activity that ranged from 10-fold to 10,000-fold above the background. The heterogeneity of lipid A fractions detected in assays with amoebocyte lysate was consistent with the finding of heterogeneity in other functional assays of lipid A fractions.     

Recognition and Treatment of Nongonococcal Urethritis in Clinical Practice

Nongonococcal urethritis is a relatively common disorder in sexually active individuals. The incidence is almost as high, if not higher, than gonorrhea. This syndrome may present with signs and symptoms indistinguishable from acute gonococcal urethritis. It is essential to differentiate the two diseases, as treatment protocols are different. Early recognition of nongonococcal urethritis and proper therapy will often lead to complete resolution and prevention of annoying complications.     

Dissociation of endotoxic activities in a chemically synthesized lipid A precursor after acetylation.

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.     

Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay.

The Limulus amoebocyte lysate endotoxin assay was evaluated as a method for rapid diagnosis of acute bacterial meningitis in a series of 305 patients. The results of Limulus assays on cerebrospinal fluid (CSF) samples from these patients were compared with the results for each patient of routine bacterial cultures and Gram stains. Positive Limulus tests were obtained on initial CSF specimens from 84% of patients with culture-proven bacterial meningitis, including all patients with meningitis due to gram-negative organisms. Initial Gram-stained smears revealed the presence of organisms in 68% of the patients. One patient with pneumococcal meningitis had a weakly positive Limulus assay, whereas patients with meningitis due to other gram-positive organisms, those with aseptic meningitis, or patients without meningitis had negative CSF Limulus tests. The Limulus assay also demonstrated the persistence of endotoxin in the CSF of certain patients during antibiotic therapy, especially patients with Haemophilus influenzae meningitis. The Limulus test proved to be a rapid, reliable indicator of the presence of gram-negative organisms in the CSF of patients suspected of acute bacterial meningitis.