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The purpose of this study was to evaluate the L-CLONE Legionella pneumophila Serogroup 1 Urine Antigen Latex Test (Access Medical Systems, Inc., Branford, Conn.) for detection of Legionella antigen in urine. A total of 481 frozen urine samples previously tested by an in-house solid-phase radioimmunoassay (RIA) was thawed and retested by using L-CLONE. Included in this sample were 140 RIA-positive samples from culture-positive or serologically confirmed cases of legionellosis and 341 RIA-negative samples from patients with non-Legionella respiratory disease or bacteriuria. The original RIA test result was accepted as the true value. L-CLONE correctly identified 76 of 140 (54%) known positive samples. False-negative results could not be attributed to a low Legionella antigen concentration or to a Legionella antigen subgroup. L-CLONE correctly identified 252 of 341 (74%) known negative samples. False-positive results were experienced in all groups of negative samples, regardless of the patients' underlying diseases. A total of 141 fresh urine samples was tested; all were Legionella antigen negative by RIA. L-CLONE provided 86% specificity. The sensitivity of the L-CLONE in testing fresh urine samples could not be evaluated because of the lack of Legionella antigen RIA-positive samples.

A patient accidentally received approximately 3 g of ofloxacin intravenously. She experienced only moderately severe central nervous system symptoms, which resolved within 9 h. The peak serum ofloxacin level was 39.3 micrograms/ml, approximately seven times the usual peak level. Ofloxacin may possess a considerable safety margin in humans.

An enzyme linked immunosorbent assay was developed to detect urinary antigen excreted by patients with Legionnaires' disease. Of 47 patients tested, antigen was detected in 39. Antigen was not detected in any of 178 urine specimens from patients with other pulmonary, bacteremic, or urinary tract infections after performance of a quick and simple confirmatory test. The assay required more time to perform than a previously described radioimmunoassay but was of equivalent sensitivity and specificity and did not require expensive equipment of contact with radioactive reagents. We conclude that enzyme linked immunosorbent assay is a rapid, sensitive, and specific means for rapidly diagnosing Legionnaires' disease which can be performed in clinical laboratories unwilling or unable to use radioisotopes.

It has been difficult to obtain pure Pneumocystis carinii antigen either from cultures or from infected lungs for use in producing a specific antibody against P. carinii. This report describes an approach toward producing a monoclonal antibody that bypasses the antigen purification steps. P. carinii infection was developed in Sprague-Dawley rats by the method of immunosuppression with cortisone. The infected lungs were homogenized, and the homogenate was used to immunize Sprague-Dawley rats. Rat spleen cells were then fused with SP2/0 mouse myeloma cells. Hybridoma clones were screened for antibody production against P. carinii by immunoperoxidase staining techniques and by enzyme-linked immunosorbent assay, using as antigens homogenates of normal rat lung, homogenates of P. carinii-infected rat lung, and harvests of P. carinii grown with WI-38 cells. Out of six hybridoma clones obtained that produced antibodies against P. carinii, one was able to produce ascitic fluid. This monoclonal antibody reacted with two P. carinii antigens with masses of about 35,000 and 65,000 daltons in P. carinii-infected lungs and three proteins with masses of about 35,000, 65,000, and 110,000 daltons in P. carinii that was harvested from a WI-38 cell culture.

Of patients with staphylococcal infections, high levels of IgM antibodies of staphylococci measured by radioimmunoassay were present in 13 of 17 patients with endocarditis, nine of 23 with complicated bacteremia, one of 20 with uncomplicated bacteremia, and two of 21 with nonbacteremic infections. Of control subjects, high levels of IgM antibodies were present in seven of 26 individuals with gram-positive infections, two of 16 with gram-negative infections, two of 20 with rheumatoid arthritis, and two of 50 uninfected persons. Concomitant elevation of IgG and IgM antibody levels occurred in 20 of 40 patients with endocarditis or complicated bacteremia compared with two of 41 patients with other types of staphylococcal infections and three of 112 control subjects. IgM antibodies only were present early in the infection in six of 13 patients with staphylococcal endocarditis or complicated bacteremia. IgG or IgM antibody levels remained elevated at least four weeks after initiation of treatment.

A radioimmunoassay for staphylococcal antigen that had detected antigenemia in each of 12 nonimmune rabbits with staphylococcal endocarditis detected antigen in sera from one of nine humans and two of eight immune rabbits with Staphylococcus aureus endocarditis. Staphylococcal antigens could be detected at concentrations as low as 0.78 microgram/ml when diluted in normal rabbit serum, compared with 6.25 microgram/ml when diluted in normal human serum and greater than 25 microgram/ml when diluted in immune rabbit or human serum. Low titers of staphylococcal antibody were found in normal rabbit serum compared with immune rabbit and normal or immune human sera. Detection of staphylococcal antigen was inhibited when the antigen was diluted in the IgG and IgM fractions, but not in the albumin fraction, of normal human serum. This study demonstrated that antigenemia can be detected infrequently in patients and immune rabbits with staphylococcal endocarditis; staphylococcal antibodies inhibit detection of antigen, presumably through formation of immune complexes.

The purpose of this study was to determine whether measurement of immunoglobulin M (IgM) antibodies against Legionella pneumophila serogroup 1 can aid in the diagnosis of Legionnaires disease. On the basis of measurements of antibody levels in 1,942 control sera, we used an IgM titer of 1:256, observed in 2.3% of the controls, as presumptive evidence of Legionnaires disease. Measurement of IgM titers permitted us to presumptively or definitively diagnose Legionnaires disease in 13 of 34 patients earlier than we would have if only IgG titers had been measured. Of the 13 patients, 5 were diagnosed serologically only by IgM antibody determination. IgM titers were presumptively diagnostic in week 1 of clinical symptoms in 4 of the 13 patients. We conclude that conjugates used for antilegionella indirect fluorescent-antibody tests should be capable of detecting IgM antibodies so that the value of serological results in diagnosing and managing Legionnaires disease will be maximized.

The purposes of this study were to determine whether antigen is excreted by patients with Legionnaires disease early enough after the onset of symptoms to be useful for making therapeutic decisions and whether antigen excretion ends when successful treatment is concluded. Specific antigen was detected in the urine of 14 (88%) of 16 patients with Legionnaires disease during days 1 to 3 of symptoms, 33 (80%) of 41 patients during days 4 to 7, 25 (89%) of 28 patients during days 8 to 14, and 11 of 11 patients after day 14, by solid-phase immunoassays for serogroup 1 Legionella pneumophila antigen. Antigen excretion persisted for 42 days or longer after the onset of treatment in at least 15 patients. The longest documented duration of excretion was 326 days. We conclude that antigen can be detected approximately as often early after symptoms begin as later, allowing meaningful therapeutic decisions to be made, but that prolonged antigen excretion may negate the diagnostic value of urinary antigen detection for relapsing or recurrent L. pneumophila pneumonia.

Eighty healthy men with nasal carriage of coagulase-positive staphylococci were given cloxacillin, rifampin, cloxacillin plus rifampin, or no drug. Treatment with rifampin alone or with rifampin plus cloxacillin eradicated the original strain from 80% of the men for 12 weeks after treatment; 15%-20% of the 80 subjects acquired a strain of a different phage type. Treatment with cloxacillin alone did not significantly alter the rate of carriage. Only one of 40 men receiving rifampin (either alone or with cloxacillin) acquired rifampin-resistant, coagulase-positive staphylococci; seven of the 40 acquired rifampin-resistant, coagulase-negative staphylococci. Rifampin-resistant organisms did not persist in repeat nasal cultures five months after treatment. Of the 40 subjects given rifampin alone or rifampin plus cloxacillin, 60% had repeat nasal cultures done one year later. Only 50% were found to be carriers of coagulase-positive staphylococci. In another study, coagulase-positive staphylococci were eradicated from 70% of 20 carriers undergoing renal dialysis who received rifampin plus cloxacillin.

A solid-phase radioimmunoassay was developed to detect antigens of Legionella pneumophila serogroup 4. The assay detected antigen in urine from four of seven patients with pneumonia caused by L. pneumophila serogroup 4, two of two patients with L. pneumophila Leiden 1 strain (proposed new serogroup), and 26 of 55 patients with L. pneumophila serogroup 1 (proven by culture or direct fluorescent-antibody assay). The antigen of two patients with serogroup 4, one with Leiden 1 strain, and 49 with serogroup 1 infections could be detected in a previously described assay for antigens of L. pneumophila serogroup 1. Serogroup specificity of the assays could be demonstrated if serial dilutions of urine were tested. None of 347 urine specimens used as controls were positive in the radioimmunoassay. We conclude that antigens are excreted in the urine of at least some patients with Legionnaires' disease caused by serogroup 4 and Leiden 1 strain. The urinary antigens in patients with serogroup 4 and Leiden 1 strain infections, although different from those in patients with serogroup 1 infections, serologically contain cross-reactive components.

Levels of antibodies to peptidoglycan were measured by solid-phase radioimmunoassay in 76 patients with Staphylococcus aureus infections and 44 patients with infections caused by other bacteria. Levels of IgM antibodies to peptidoglycan were elevated in 24% of the patients with endocarditis or complicated bacteremia caused by S. aureus, 21% of those with serious infections caused by other gram-positive cocci, none of those with less serious infections caused by S. aureus or gram-negative bacilli, and 5% of the normal controls. Levels of IgG antibodies to peptidoglycan were elevated in 50% of the patients with endocarditis or complicated bacteremia caused by S. aureus, 3% of those with less severe infections caused by S. aureus, 17% of those with infections caused by other gram-positive cocci, 20% of those infections caused by gram-negative bacilli, and 5% of the normal controls.

Specific antibodies have previously been found to impair detection of staphylococcal antigen by solid-phase radioimmunoassay. The inhibitory effect of heat-labile serum factors on the detection of antigen is now described. Heating of serum at 56 C for 30 min prior to addition of antigens of Pseudomonas aeruginosa or Staphylococcus aureus improved sensitivity by eight- to 32-fold. Heating was less effective when performed after addition of antigen. Treatment with zymosan reduced the inhibitory effect of serum, although less effectively than did preheating. Antigen in buffer was detected with 16 times less sensitivity in antibody-coated tubes exposed to fresh serum than in tubes exposed to heated serum. These findings suggest that complement factors can interact with antibody in coated tubes and at least some antigens and thereby inhibit detection of antigen. The finding that heat treatment improved the sensitivity of Ausria II -125 (Abbott Laboratories, North Chicago, Illinois) for hepatitis B surface antigen is of potential immediate clinical applicability.

To aid in the rapid diagnosis of staphylococcal infection, a solid-phase radioimmunoassay for Staphylococcus aureus antigen was developed and evaluated in rabbits with staphylococcal endocarditis. Test specimens containing antigen were added to polystyrene tubes coated with antibody to S. aureus. Antigens immobilized on the tube were detected by adding radiolabeled antibody to S. aureus. Sensitivity for antigen was 0.312 microgram/ml in buffer and 1.25 microgram/ml in 50% rabbit serum. Cross-reactions were not observed with antigens extracted from streptococci; however, antigen extracted from Staphylococcus epidermidis (which contained ribitol-teichoic acid) could also be detected at low concentrations. Antigen was detected in each of 12 rabbits with S. aureus endocarditis but not in control rabbits. This assay is sensitive, specific, reproducible, and capable of detecting antigens in the serum of rabbits with endocarditis.

Detection of a Histoplasma capsulatum polysaccharide antigen (HPA) has proved a useful approach to diagnosis of histoplasmosis. Two sandwich enzyme-linked immunosorbent assays (ELISAs) using alkaline phosphatase (AP)-or horseradish peroxidase (HRP)-conjugated antibodies were compared with solid-phase radioimmunoassay (RIA) for detection of HPA. The AP-ELISA and HRP-ELISA were each positive in 17 (89.5%) of 19 urine specimens from patients with disseminated histoplasmosis, while the RIA was positive in 18 (94.7%) of 19. Specimens from patients with nondisseminated histoplasmosis were positive by AP-ELISA in 8 of 32, by HRP-ELISA in 4 of 25, and by RIA in 12 of 25. Of control specimens from patients with other fungal infections, the AP-ELISA was negative in 22 (91.7%) of 24, the HRP-ELISA in 23 (95.8%) of 24, and the RIA in 23 (95.8%) of 24. Reproducibilities AP-ELISA and HRP-ELISA were 95.1% and 95.1%, respectively. Thus, AP-ELISA and HRP-ELISA appear less sensitive than RIA and may be falsely negative in specimens containing low levels of HPA.

We assessed the ability of a solid-phase radioimmunoassay, modified to allow antigen detection in serum, to detect circulating antigens in granulocytopenic rabbits with Pseudomonas aeruginosa bacteremia. In vitro experiments with Pseudomonas lipopolysaccharide indicated that treatment of serum-lipopolysaccharide mixtures with heat, chloroform, or heparin improved the sensitivity for detecting lipopolysaccharide 8- to 16-fold. Chloroform treatment permitted antigen detection in serum or plasma of bacteremic rabbits in which antigen could be detected poorly or not at all in untreated specimens. However, chloroform-treated specimens occasionally caused dissolution of plastic tubes, resulting in nonspecific binding of 125I-labeled anti-Pseudomonas immunoglobulin G. Heating sera at 56 degree C for 30 min improved antigen detection both in both in lipopolysaccharide-serum mixtures and in bacteremic rabbits. Antigen was detected in the heated serum or plasma of 20% of 20 culture-positive granulocytopenic rabbits, none of 15 culture-negative granulocytopenic rabbits, and none of 38 normal rabbits. Antigen was detected in none of 15 rabbits with 2 to 300 colony-forming units of P. aeruginosa per ml of blood and in 4 of 5 rabbits with > 10(3) colony-forming units per ml. We conclude that circulating antigens are present in the blood of rabbits with high-level P. aeruginosa bacteremia and that these antigens can be detected by solid-phase radioimmunoassay. Further improvements in assay sensitivity will be required to detect antigens, if present, in animals with lower levels of P. aeruginosa bacteremia.

We describe a simple and rapid method to dissociate antigen-antibody complexes to allow antigen detection by radioimmunoassay. Immunoglobulins and immune complexes were precipitated from serum in 50%-saturated ammonium sulfate. The precipitate was dissolved in distilled water and heated at 90 degrees C for 15 min to free the antigen and denature the antibody irreversibly. With this treatment, the sensitivity for detecting staphylococcal antigen added to human serum improved greater than or equal to 16-fold and was similar to the sensitivity in buffer. After intravenous injection of antigen into a rabbit with high levels of staphylococcal antibodies, antigen was detected only after thermodissociation. Also, in 4 of 26 patients with Staphylococcus aureus bacteremia, circulating antigen was detected after thermodissociation. Thermodissociation of immune complexes is a simple, rapid method and should be useful for detecting other heat-stable antigens.

We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically.

A solid-phase radioimmunoassay designed to detect serotype 6 Pseudomonas aeruginosa antigens was evaluated for its ability to rapidly diagnose urinary tract infections. Twelve P. aeruginosa serotypes were easily differentiated in the assay from eight other gram-negative bacterial species. During log-phase growth, the assay detected antigens in culture when approximately 10(6) or more serotype 6 P. aeruginosa organisms were present. Both cell-associated and solubilized antigens were detected. The assay detected antigens in 13 of 17 urine specimens which grew greater than 10(5) P. aeruginosa, 3 of 38 which grew other gram-negative rods, and none of 83 with no growth. Two of the three positive specimens from the other gram-negative rod group probably also contained P. aeruginosa. No preincubation of the urine specimens was required, and results were available within 2.5 h. The assay represents an improvement over other procedures for rapidly diagnosing urinary tract infections in that it allows diagnosis by species and should be adaptable to semiautomation.

Because the reported frequency of teichoic acid precipitins in controls and various patient groups has varied considerably among laboratories, we studied the effect of using various concentrations of staphylococcal extracts in agar-gel diffusion tests for teichoic acid antibodies. Of 25 normal sera, only 1 was positive against an undiluted extract, but 4 were positive against a 16-fold-diluted extract. Of nine sera from patients with staphylococcal bacteremia, two were positive at a higher titer against the diluted extract. A false-positive serum against the undiluted extract had a twofold titer increase against the diluted extract. Because human immune serum globulin is generally used as a positive teichoic acid antibody control, the variability of five different lots was studied. Three lots ahd teichoic acid antibody titers of 1:4, whereas one each had titers of 1:2 and 1:8. Based on this study, we feel that staphylococcal extracts should not be diluted. If immune serum globulins are used to determine the adequacy of ultrasonic extracts, newly acquired globulin lots should be standarized against an ultrasonic extract of proven sensitivity and specificity.