Rapid presumptive bacteriological diagnosis of Legionnaires disease.

A simple, relatively rapid silver impregnation stain has been found to stain Legionella pneumophila effectively in paraffin-embedded tissue sections while permitting visualization of histological detail. It may also be used to stain the organism in body fluids. The stain is not specific and thus must be confirmed by direct fluorescent-antibody technique or culture, but, in the absence of other bacilli demonstrable by Gram or other stain, visualization of typical bacillary forms in a patient with illness compatible with Legionnaires disease provides strong presumptive evidence supporting this diagnosis.     

Immunoglobulin M antibody capture enzyme-linked immunosorbent assay for diagnosis of St. Louis encephalitis

Sera from patients with St. Louis encephalitis were tested with an immunoglobulin M (IgM) antibody capture enzyme immunoassay (MAC ELISA). The assay used five reagents: antihuman IgM, test serum, sucrose-acetone-extracted mouse brain antigen, broadly cross-reactive flavivirus monoclonal antibody conjugated to alkaline phosphatase, and substrate (p-nitrophenyl phosphate). MAC ELISA endpoint titers correlated (r = 0.893) with the absorbance value of a 1:100 dilution of patient serum. Significant (fourfold or greater) changes in the endpoint titers between paired sera corresponded to a critical ratio (ratio of absorbance values at the 1:100 dilution) of greater than or equal to 1.3. IgM antibodies were detected in 71% of patients bled at 0 to 3 days after the onset of illness, in 99% bled at 4 to 21 days, and in 91% bled at 22 to 67 days. Thereafter, the IgM seropositivity rate declined; however, 29% of sera were still positive at 115 to 251 days after the onset of illness. MAC ELISA titers were significantly correlated with hemagglutination inhibition (r = 0.258) and neutralization (r = 0.711) titers. Because IgM antibodies appeared early and waned rapidly, a diagnosis was made on the basis of a decrease in titer more often by MAC ELISA than by hemagglutination inhibition, complement fixation, or neutralization tests. IgM antibodies generally showed a high degree of specificity; heterologous cross-reactions were, however, present in 4 of 14 sera examined. The MAC ELISA is useful for the rapid, early diagnosis of St. Louis encephalitis.     

Endomysial antibodies in the diagnosis of celiac disease and the effect of gluten on antibody titers

Celiac disease, a common chronic gastrointestinal disorder, is gluten induced and is controlled with a gluten-free diet. While the management of CD with a gluten-free diet is quite effective, the diagnosis is rather difficult. The ESPGAN criteria for the diagnosis of CD seems to be tedious and time-consuming. Serological tests for IgA class endomysial antibodies, as detected by indirect immunofluorescence, on human and primate smooth muscles are specific and sensitive markers of celiac disease. Of all the specimens examined, endomysial antibodies were present in patients with gluten-sensitive enteropathy. These antibodies occurred in all active cases of celiac disease, in 90 percent suspected celiac patients where all the ESPGAN criteria has not been fulfilled. This contrasts to the presence of endomysial antibodies in 46 percent of confirmed and 17 percent of suspected celiac patients maintained on a gluten-free diet for various time intervals. Endomysial antibodies also occurred in all cases with chronic diarrhea and gut histology consistent with CD and 8% of asymptomatic family members of CD patients. None of the patients with other gastrointestinal and liver diseases had endomysial antibodies. These studies thus emphasize the specificity and sensitivity of endomysial antibodies for celiac disease.     

Effect of various histological fixatives on fluorescent antibody detection of Legionnaires disease bacteria.

Human lung tissue containing the Legionnaires disease bacterium was fixed in seven different histological fixatives, processed, and embedded in paraffin. Deparaffined sections from each were stained by fluorescent antibody and by Dieterle silver impregnation. With the fluorescent antibody stain, the Legionnaires disease bacterium could be detected in tissues prepared with any of the fixatures, but the Dieterle silver impregnation was not satisfactory on Zenker-fixed tissues.     

Legionnaires disease: historical perspective.

In the summer of 1976, a mysterious epidemic of fatal respiratory disease in Philadelphia launched an intensive investigation that resulted in the definition of a new family of pathogenic bacteria, the Legionellaceae. In retrospect, members of the family had been isolated from clinical specimens as early as 1943. Unsolved epidemics of acute respiratory disease dating to the 1950s were subsequently attributed to the newly described pathogens. In the intervening years, the Legionellaceae have been firmly established as important causes of sporadic and epidemic respiratory disease. The sources of the infecting bacteria are environmental, and geographic variation in the frequency of infection has been documented. Airborne dissemination of bacteria from cooling towers and evaporative condensers has been responsible for some epidemics, but potable water systems are perhaps more important sources. The mode of transmission from drinking water is unclear. The Legionellaceae are gram-negative, facultative, intracellular pathogens. The resident alveolar macrophage, usually an effective antibacterial defense, is the primary site of growth. Cell-mediated immunity appears to be the most important immunological defense; the role of humoral immunity is less clear. Erythromycin remains the antibiotic of choice for therapy of infected patients, but identification and eradication of environmental sources are also essential for the control of infection.     

Identification of diaminopimelic acid in the Legionnaires disease bacterium.

Diaminopimelic acid was found to be a component of the cell wall of the Legionnaires disease bacterium, thus providing additional evidence that the organism is a bacterium. The presence of this amino acid was determined by gas-liquid chromatography and confirmed by gas chromatography-mass spectrometry.     

Exotoxin activity associated with the Legionnaires disease bacterium.

Hemolysis occurred around growth of the Legionnaires disease bacterium on supplemented Mueller-Hinton agar containing sterile defibrinated blood from each of five mammalian species. Hemolysis was most pronounced with guinea pig or rabbit blood, was less intense with horse or sheep blood, and was slight with blood from a human donor. Sterile filtrates of allantoic fluid from embryonated hen's eggs that had been infected with this organism displayed hemolytic activity in a radial hemolysis assay with guinea pig cells in agar. Growth of the Legionnaires disease bacterium on F-G agar with 5% hen's egg yolk was surrounded by a zone of clearing and more circumscribed zones of iridescence and increased opacity on and in the medium. Attempts to detect activity analogous to that of Escherichia coli heat-labile or heat-stable enterotoxin in allantoic fluid from infected eggs or in cultures of the Legionnaires disease bacterium were not successful.     

Utility of Leu M1 monoclonal antibody in the differential diagnosis of Hodgkin's disease

We conducted a retrospective study of the utility of Leu M1 monoclonal antibody staining of paraffin-embedded tissue in the differential diagnosis of Hodgkin's disease (HD). Forty-two cases of HD of various histologic types and 33 cases of non-HD lymphomas and hyperplasias were stained with Leu M1 using the avidin-biotin-peroxidase complex technique. Varying numbers, but not all Reed-Sternberg (RS) and Hodgkin's cells in all 42 cases of HD, were Leu M1 positive. These cases included seven examples of interfollicular HD and nine cases of lymphocyte-predominance HD, eight of which were nodular. Four of five cases of immunologically proved T-cell lymphoma contained Leu M1-positive RS-like cells, and Leu M1-positive RS-like cells were noted in two of five cases of non-HD lymphoma that were not phenotyped but were morphologically consistent with T-cell lymphoma. We concluded that Leu M1 staining is an aid in the diagnosis of HD, but it cannot be used to differentiate HD from T-cell lymphoma containing RS-like cells.     

Primary isolation media for Legionnaires disease bacterium.

Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) were plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% Iso Vitale X and 1% hemoglobin (MH-IH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the Iso Vitale X reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different cultures conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35 degrees C under 2.5% CO2 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH.     

Immunoglobulin M antibody response against Mycoplasma pneumoniae lipid antigen in patients with acute pancreatitis.

Serial serum samples from patients with acute pancreatitis showed a significant increase in antibodies against methanol-chloroform-extracted lipid antigen from Mycoplasma pneumoniae when tested by complement fixation. The antibodies did not react with antigens prepared from other human mycoplasmas or from pancreatic tissue by lipid extraction. The antibodies were predominantly immunoglobulin M (IgM). No correlation with cold agglutinins or cardiolipid complement-fixing antibodies was found. The IgM antibody response seemed to be prolonged: after 3 to 4 weeks the antibodies were still in many cases exclusively IgM. Similar IgM responses were also found in certain cases of acute meningoencephalitis. We postulate that during the disease antigenic components identical or very similar to major determinants in the M. pneumoniae lipid antigen are revealed and elicit the IgM antibody response. Their resemblance to natural antibodies and their possible biological role is discussed.     

Onset and duration of urinary antigen excretion in Legionnaires disease.

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.     

Cellular fatty acid composition of isolates from Legionnaires disease.

The cellular fatty acids of four isolates from Legionnaires disease and two antigenically related isolates were identified by gas chromatography, mass spectrometry, and associated techniques. The six isolates had essentially the same fatty acid composition, which was characterized by large amounts (greater than 80%) of branched-chain acids.     

Immunoglobulin M in Murray Valley encephalitis.

Twelve clinical cases of Murray Valley encephalitis are described, in which the sero-diagnosis was confirmed by the detection of Murray Valley encephalitis immunoglobulin M.     

Recognition of a new serogroup of Legionnaires disease bacterium.

A strain of the Legionnaires disease bacterium (LDB) that was isolated by Joseph E. McDade from a postmortem lung specimen of a patient with fatal atypical pneumonia at the Veterans Administration Hospital in Togus, Maine was serologically different from 16 other strains of LDB that had been isolated previously from patients in other geographic locations. The serological differences of the Togus isolate were shown in results of direct and indirect fluorescent antibody staining and of immunoelectrophoresis with soluble antigen extracts. Seroconversion for the Togus strain of LDB in acute- and convalescent-phase sera from a second patient with atypical pneumonia at the Veterans Administration Hospital in Togus indicated that this patient had been infected with an LDB that was serologically similar or identical to the Togus isolate. The Togus serogroup of LDB should be considered when performing serological tests for Legionnaires disease.     

Detection of Legionnaires disease bacteria by direct immunofluorescent staining.

Antisera and fluorescein isothiocyanate conjugates prepared for five strains of the Legionnaires bacteria were tested in both homologous and heterologous staining reactions with 10 isolates of the organism from patients in seven geographic areas. The strains were related but not identical as judged by the results of direct immunofluorescence staining. The conjugates were successfully used to detect Legionnaires disease bacteria in Formalin-fixed lung scrapings, in histological sections, and in fresh lung tissue obtained at biopsy or autopsy. In addition, the labeled antibodies are valuable for staining suspected cultures of the bacterium and for searching for the source of these organisms in soil, water, and other environmental niches. The reagents are highly specific for detecting the Legionnaires organism in clinical specimens.     

Immunoglobulin G and M composition of naturally occurring antibody to type III group B streptococci.

Human sera were examined by an enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgM antibodies to purified type III polysaccharide of group B streptococci. The antigen-binding capacity of a reference human serum was determined by a radioimmunoassay, and the total antibody content was determined by quantitative precipitation. The serum was then depleted of IgM and IgA to determine the effect on the antigen-binding capacity. Duplicate samples of 81 sera were tested by the enzyme-linked assay in comparison with reference standard serum. Although levels of IgG antibody were greater in subjects who had carried type III streptococci during pregnancy, concentrations of this antibody were generally low. Only 2 of 28 sera (7%) from parturient subjects and 7 of 25 sera (28%) from adult volunteers contained greater than or equal to 1 microgram of IgG antibody per ml; the mean levels were 0.13 and 0.53 micrograms/ml, respectively. In contrast, 19 of 28 maternal sera (68%) and 22 of 25 (88%) volunteer adult sera contained greater than or equal to 1 microgram/ml of IgM antibody; mean levels were 1.33 and 1.54 micrograms/ml, respectively. The cord serum levels of IgG antibody were almost identical to maternal serum concentrations, whereas IgM antibody was essentially undetected.     

Further studies of the cellular fatty acid composition of Legionnaires disease bacteria.

The cellular fatty acid composition of 36 strains of the Legionnaires disease bacterium was determined by gas chromatography after growth on different media. The fatty acid profile of each strain was essentially identical on each medium and was characterized by large amounts (greater than 68%) of branched-chain acids.     

Serology of Legionnaires disease: comparison of indirect fluorescent antibody, immune adherence hemagglutination, and indirect hemagglutination tests.

An immune adherence hemagglutination (IAHA) test for the measurement of antibodies to Legionella pneumophila was developed and evaluated for the diagnosis of Legionnaires disease. Its sensitivity was compared to that of the indirect fluorescent antibody (IFA) test and a recently developed indirect hemagglutination (IHA) test. The sensitivity of the three tests appeared to be similar, with the IFA test giving slightly higher titers. Both the IHA and IAHA tests appear useful for the serodiagnosis of Legionnaires disease; the IAHA test has the advantage that it can be used with many other serological antigens.