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.     

Application of a Limulus test device in rapid evaluation of gonococcal and nongonococcal urethritis in males

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.     

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.     

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.     

New, sensitive rocket immunoelectrophoretic assay for measurement of the reaction between endotoxin and Limulus amoebocyte lysate.

To study the active proteins which participate in the reaction of Limulus amoebocyte lysate (LAL) with lipopolysaccharide, antibody was raised in rabbits against LAL. When LAL was run against rabbit antiserum in crossed immunoelectrophoresis, a complex precipitin pattern appeared. Profound changes took place after reaction of LAL with lipopolysaccharide. The most distinct change was the complete disappearance of the cathodic migrating protein coagulogen, because the antigenicity of coagulogen was lost. Based on this observation, a new rocket immunoelectrophoretic method was developed to detect the disappearance of coagulogen after reaction of LAL with lipopolysaccharide. This assay method was used on clinical specimens (cerebrospinal fluid, plasma, ascites, and urine). It was used as a qualitative test, when a single sample is tested, or as a quantitative assay, when a number of sample dilutions were tested. The new method showed a higher degree of accuracy and sensitivity in comparison with the tube test and it can be used for both research and diagnostic purposes.     

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.     

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.     

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.     

Ability of gonococcal and meningococcal lipooligosaccharides to clot Limulus amebocyte lysate.

We investigated whether the striking difference in severity of coagulopathy observed between bacterial sepsis involving Neisseria meningitidis and Neisseria gonorrhoeae species is related to species-dependent abilities to directly activate coagulation. Using lipooligosaccharide (LOS)-activated gelation of Limulus amebocyte lysate, we compared the relative abilities of outer membrane LOS of 10 N. meningitidis and 10 N. gonorrhoeae strains to initiate coagulation. A wide range of procoagulant potencies was observed for each species, and there was significant overlap of potencies between species. Relative biological activities did not correlate with the oligosaccharide components as defined by LOS molecular weight or specific antigenic epitopes. Purified lipid A of two LOS strains of different potency demonstrated relative procoagulant biological activities similar to those of their parent LOSs. When these lipid A preparations were further separated by thin-layer chromatography, the most polar component of each lipid A possessed the majority of the procoagulant activity. We concluded that the ability of neisserial LOS to initiate coagulation of Limulus lysate is a property of the lipid A portion of the molecule and is most likely determined by fine structural differences in the lipid A which are independent of species.     

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.     

Microbial population diversity in the urethras of healthy males and males suffering from nonchlamydial,nongonococcal urethritis

Nonchlamydial, nongonococcal urethritis (NCNGU) is suggested to be a sexually transmitted disease in men. NCNGU patients were compared to control subjects with regard to the presence of potentially infectious bacteria in the first void urine. Patients' pre- and post-antibiotic-treatment urine samples and two samples obtained 2 weeks apart from healthy volunteers, who did not receive antibiotic therapy, were analyzed with broad-spectrum PCR tests aiming at eubacterial small subunit rRNA genes. Restriction fragment length polymorphism analysis of the amplicons cloned from the mixtures of PCR products revealed that many different species of microorganisms were found to be colonizing the male urethra. We document here clear differences in the composition of the resident urethral flora between samples obtained from various individuals and between samples obtained at various points in time for a single individual. No major changes in population complexity were found upon antimicrobial treatment. In two of five patients a previously suggested pathogen (Mycoplasma genitalium or Haemophilus parainfluenzae) was accurately identified on the basis of DNA sequencing. No ubiquitous, azithromycin-sensitive organism was identified as a common pathogen in all patients, but up to 40% of all clones represented as-yet-unclassified bacterial species. Relatively often Pseudomonas spp. or Pseudomonas-like organisms were identified in the bacterial flora of patients. Interestingly, an as-yet-uncharacterized microbial species was identified as a negative predictor of NCNGU. This species was identified in all control subjects and was absent from all of the patient' samples (5 of 5 versus 0 of 5, P = 0.0079). This suggests that NCNGU might also be diagnosed by assessing the absence rather than the presence of certain bacterial species.     

Evaluation of absorption with Limulus amebocyte lysate to remove contaminating endotoxin from interferon and lymphokine preparations

Alpha and beta human interferon (IFN) preparations and lymphokines (supernatants of PHA-stimulated blood lymphocytes) were deliberately contaminated with endotoxin (20 ng/ml) and subsequently rendered endotoxin-free by absorption with Limulus amebocyte lysate (LAL). Absorption with LAL did not appreciably affect the antiviral activity of IFN and lymphokines in 8 experiments and caused a 30-50% reduction in two. The capacity of these agents to stimulate natural killer cell activity and monocyte cytotoxicity was not consistently modified by absorption on LAL. When the chemotactic activity of lymphokine for monocytes was measured, the maximal number of monocytes induced to migrate and the maximal active lymphokine concentration were not affected by absorption with LAL. LAL-treated lymphokines, however, showed a prozone phenomenon, presumably related to the release of chemotaxis inhibitor(s) from the LAL gel.     

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.     

Localization of gonococcal lipopolysaccharide and its relationship to toxic damage in human fallopian tube mucosa.

An experimental model using human fallopian tubes in organ culture was used to study the localization of purified gonococcal lipopolysaccharide (LPS). LPS was visualized by light microscopy with immunoperoxidase staining. Immediately after addition to fallopian tube organ cultures, gonococcal LPS aggregated on the tips of cilia. By 1 to 2 h after exposure, LPS could be seen distributed throughout the cytoplasm of ciliated and nonciliated cells in structures resembling vesicles. By 12 h, there were sloughed, ciliated cells present in the fallopian tube lumen, which had positive LPS stain on their surfaces as well as in their cytoplasm. By 24 h, LPS was distributed throughout the cytoplasm. Control experiments with rabbit oviduct organ cultures showed that LPS failed to attach, enter, or damage mucosal cells. These studies illustrate the initial localization of LPS on human mucosal cells and its uptake into the cells, which are coincident with toxicity for ciliated epithelial cells.     

Purification and characterization of an allergen from celery immunochemically related to an allergen present in several other plant species. Identification as a profilin

The purification of a 15 kD allergen from celery was obtained by a four step procedure. Evidence for at least two isoallergenic forms was obtained after analysis by two-dimensional-electrophoresis. A rabbit polyclonal antibody raised against this purified allergen allowed us to confirm our precedent results on the occurrence of allergenically and molecular mass-related components in celery and birch and mugwort pollens. In addition such components were also present in numerous other species like Cynodon dactylon, Sorghum halopense, Poa pratensis, Ambrosia elatior and in apple and carrot. The 15 kD allergen was identified as profilin by use of a specific rabbit polyclonal antibody that recognized a recombinant birch profilin. In addition, the purified celery allergen binds IgE from sera of patients allergic to birch profilin. These results reinforce the concept of profilin as a panallergen responsible in some patients for cross-allergic manifestations to various and unrelated species of grasses, weeds, trees, vegetables and fruits.     

Direct induction of tissue factor synthesis by endotoxin in human macrophages from diverse anatomical sites.

On exposure to endotoxin and other stimuli, human peripheral-blood mononuclear cells generate a potent procoagulant activity (PCA), identified as tissue factor. Although it is now recognized that the monocytes are the source of PCA, the question whether these cells per se are capable of procoagulant response to endotoxin or require lymphocyte collaboration remains unsettled. We have investigated the capacity of highly purified human macrophages from diverse anatomical sites to generate PCA following endotoxin stimulation. Purified (greater than 99%) monocyte-derived macrophages were obtained by prolonged (3-10 days) in-vitro culture of adherent monocytes using medium supplemented with 50% human serum. Purified (greater than 95%) peritoneal and milk macrophages were isolated by adherence to plastic. PCA was measured before and after incubation (4 hr at 37 degrees) with endotoxin (Salmonella enteritidis LPS, W or Escherichia coli O111:B4LPS, W, 1 microgram/ml final concentration) using a one-stage clotting assay and/or a two-stage amidolytic assay. Monocyte-derived macrophages had low baseline PCA (14-19 units/10(5) cells) but, upon exposure to endotoxin, displayed an eight-fold increase in PCA over control. Peritoneal and milk macrophages expressed very low baseline activity (1-5 units/10(5) cells). The latter, however, increased 15-20 times over control following endotoxin stimulation. PCA was identified as tissue factor by biological and immunological criteria. Its generation was completely abolished by cycloheximide. It is concluded that in the human mononuclear phagocyte series the capacity to produce PCA is not restricted to circulating monocytes but is also expressed by macrophages obtained from diverse anatomical sites. These macrophages appear to be autonomous in their procoagulant response to endotoxin.