Pediatric Legionnaires' disease: diagnosis by direct immunofluorescent staining of sputum.

Legionnaires' disease occurred in a 3-year-old boy with Down's syndrome. His illness was characterized by bilateral pneumonia, high fever, and response to erythromycin. The diagnosis was made by demonstrating Legionella pneumophila, serogroup 1, in sputum with a direct fluorescent antibody stain. L pneumophila antigen was detected in urine by an enzyme-linked immunospecific assay. The diagnosis was confirmed by a more than fourfold rise in serum antibody titer. Although Legionnaires' disease appears to be uncommon in children, it should be included in the differential diagnosis of pneumonia in the immunocompromised child.

Status of serologic tests for Legionella antigen and antibody at the Centers for Disease Control.

Nine species and 16 serogroups have been defined for the genus Legionella. Direct immunofluorescence assay (DFA) results with 12 conjugates in 3 polyvalent pools showed that L. pneumophila serogroup 1 comprised 63% of the legionellae identified in specimens from patients with legionellosis. Indirect immunofluorescence assay (IFA) titers of paired sera from patients with suspected legionellosis against multiple Legionella antigens showed preferential reactivity for the L. pneumophila antigens. The multiple-antigen IFA was 96% specific when sera from patients with disease due to other etiologic agents were tested. The prevalence of antibody among well individuals is relatively high in some population groups.

Immunodiagnostic tests for Lyme disease.

Standardized serologic tests for Lyme disease are needed, as isolation or in situ demonstration of the spirochete has proved difficult. At the Centers for Disease Control (CDC), an indirect immunofluorescence assay (IFA) was modified from a previously described IFA, and an enzyme-linked immunosorbent assay (ELISA) was developed with soluble spirochetal antigens. Both tests were evaluated with sera from Lyme disease patients, normal controls, and patients with other diseases. They were highly specific for Lyme disease when sera from patients with syphilis were excluded. Sensitivity varied with disease stage: for patients with erythema chronicum migrans alone, the IFA was 53 percent sensitive and the ELISA was 67 percent sensitive. In contrast, all patients with complicated Lyme disease had at least one serum specimen positive in both tests. Twenty-six percent of the sera from 289 patients with suspected Lyme disease that were submitted to CDC in 1983 had IFA titers greater than or equal to 256 and thus were considered positive. Both tests should be useful diagnostic and epidemiologic aids.

Analysis of group B streptococcal types associated with disease in human infants and adults.

It is important to resolve existing differences of opinion regarding group B streptococcal type distribution in human disease because of the relevance of type prevalence to future programs of prevention. This report compares data obtained from typing 392 group B streptococci isolated from systemic infections in both infants and adults in the United States from 1972 through 1975. The data showed a substantial predominance of type III among strains isolated from cases of infant meningitis and from "late-onset" septicemia but did not confirm a prior report that type Ia causes most cases of "early-onset" infant septicemia. Type II was the predominant serotype among 11 cerebrospinal fluid isolates from adults. The fact that over one-fourth of the isolates were types other than Ia or III means that future epidemiological studies, including definition of immunological factors, must include all five group B types.

Detection of group B streptococcal antibodies in human sera by radioimmunoassay: concentrations of type-specific antibodies in sera of adults and infants infected with group B streptococci.

Current interest in determining the possible protective role of antibodies against group B streptococcal disease prompted this study of the feasibility of using a radioimmunoassay to measure type-specific immunity in humans. The radioimmunoassay was standardized as a quantitative test for antibodies against the carbohydrate (CHO) antigens of all five group B types. The data showed that the CHO antigens extracted by a cold trichloroacetic acid-sonification method measure more antibodies than do the corresponding CHO antigens extracted by hot hydrochloric acid; that the Ia CHOs extracted from two different types, Ia and Ic, measure the same quantity of Ia antibodies; and that human sera contain antibodies reactive with all five type-specific CHOs. No evidence of "protective" antibody was found in the serum samples studied, although there was evidence of and antibody response in adults to prolonged colonization by group B streptococci. The wide ranges of antibody concentration in a serum bank collection, the broad reactivity of all human sera tested, and the mixed populations of antibodies in human sera that react with different determinants on the same type-specific CHO antigen (type III) indicate that further studies must be done to better define normal and susceptible populations and to determine antigenic components important in protection.

Immunochemistry of purified polysaccharide type antigens of group B streptococcal types Ia, Ib, and Ic.

The HCl-extracted purified polysaccharide type antigens of group B Streptococcus types Ia and Ib were composed of galactose and N-acetylglucosamine in a molar ratio of 3:1 for the Ia antigen and 2:1 for the Ib antigen. Immunochemical data were the same for the Ia antigens of type Ia, purified in this study, and type Ic, purified earlier. Glucosamine inhibited the Ib quantitative precipitin reactions more effectively than did N-acetylglucosamine, whereas the reverse was true of the Ia reaction. Ouchterlony studies were consistent with these observations and also revealed two type-specific precipitin bands with the HCl-extracted Ia antigens. All saline-extracted type antigens, however, formed single Ouchterlony bands that were only partially identical to the corresponding HCl antigens. Purification of the saline antigens was accomplished by treatment with cold trichloroacetic acid and by fractional ethanol precipitation. Immunoelectrophoresis experiments showed that the saline antigens were more negatively charged than the HCl antigens. The presence of sialic acid in the saline antigens probably accounted for their net negative charge and the fact that they were partially degraded by mild acid hydrolysis. The same serological specificities were observed with saline- and with HCl-extracted antigens.

Comparison of streptococcal R antigens.

At least four distinct nonspecific protein R antigens were found in streptococci of groups A, B, and C by immunodiffusion in agar gel with anti-R sera.

Nontypable group B streptococci isolated from human sources.

The present study was done to determine whether so-called nontypable (NT) group B streptococci from human sources possess as yet unrecognized type antigens. Antisera were raised in rabbits against several NT strains and then tested with hydrochloric acid extracts of 53 NT group B streptococci. One serum was strain specific, another was nonspecific in that it contained only R-protein antibodies, and a third (NT1), although apparently type specific, reacted with only five strains. These results do not justify using NT1 serum in the group B typing system.

CAMP-disk test for presumptive identification of group B streptococci.

The modification of the CAMP test for goup B streptococci involved substituting a paper disk impregnated with partially purified beta-hemolysin for the staphylococcal culture that was the source of beta-hemolysin in the original test. The disk is placed onto a sheep blood agar plate beside the streak of Streptococcus being tested. The plate is then incubated aerobically at 35 degrees C. A positive reaction consists of a lunar-shaped clear zone that appears within 24 h in the dark beta-hemolysin zone surrounding the disk. A double-blind study of 135 randomly coded streptococcal isolates showed complete agreement between the CAMP-disk test and the standard Lancefield precipitin test. All group B streptococci tested had positive reactions, and all strains tested from streptococcal groups A, C, D, and G were negative. The CAMP-disk test is a simple and convenient way to identify presumptively group B streptococci.

Formalin-killed versus heat-killed Legionella pneumophila serogroup 1 antigen in the indirect immunofluorescence assay for legionellosis.

Indirect immunofluorescence assay titers of human sera obtained against the Legionella pneumophila serogroup 1 antigen (Philadelphia 1 strain) killed with 10% Formalin showed a tendency to be lower than those obtained against the reference heat-killed antigen (geometric mean titer of 194 and 370, respectively) when all other variables in the test were held constant. Test results were interpreted the same for 96% of 60 paired sera if the cutoff level used to interpret a positive test result for the formalinized antigen was lowered by one twofold dilution factor.

Practical considerations in using counterimmunoelectrophoresis to identify the principal causative agents of bacterial meningitis.

Many clinical laboratories are currently using counterimmunoelectrophoresis (CIE) as an aid in the rapid diagnosis of bacterial meningitis. Because cross-reactions among causative agents have been reported, the present study was undertaken to explore the problems that might occur when reference and commercial antisera are used in CIE. Broth cultures of 35 bacterial strains were tested with 76 reference and commercial antisera by CIE. Some of the antisera tested failed to react with their homologous strains. Furthermore, several cross-reactions between genera, as well as within species, were noted. These findings suggest that precautions must be taken to insure that all materials used in CIE tests are of high quality. If properly performed and interpreted, CIE may be a valuable adjunct in the identification of organisms causing bacterial meningitis, but it is, nevertheless, a presumptive test and should not be used to replace the Gram stain and culture techniques.

Radioimmunoassay for measuring antibodies specific for group B streptococcal types Ia, Ib, Ic, II, and III.

The object of this study was to develop a test that would measure antibodies directed against group B streptococcal types Ia, Ib, Ic, II, and III. The type-specific carbohydrate antigens were purified, labeled with 125I, and used to develop a radioimmunoassay. This procedure should be particularly useful in testing human sera for group B type-specific antibodies, since it requires very small quantities of antigens and measures primary type antigen-antibody interactions.

Evaluation of a solid-phase immunofluorescence assay for detection of antibodies to Legionella pneumophila.

A semiautomated solid-phase immunofluorescence technique (FIAX) was compared with the standard indirect immunofluorescence assay (IFA) for the determination of antibody levels to Legionella pneumophila serogroup 1 in paired human serum samples. The FIAX method was in agreement with the IFA test for 91.8% of the serum pairs but gave evidence of a recent Legionella infection significantly fewer times than did the IFA. These results suggest that the FIAX technique may eventually be a useful alternative test for measuring Legionella antibodies. However, further study will be required to determine its efficacy in providing a serodiagnosis of legionellosis.

Detection of cell-associated or soluble antigens of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei by staphylococcal coagglutination tests.

Current methods used for the detection of whole-cell isolates of Legionella or for the detection of Legionella soluble antigens are technically impractical for many clinical laboratories. The purpose of this study was to explore practical alternatives. The results showed that whole cell isolates of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei were identified specifically by a simple slide agglutination test or slide coagglutination test in which the reagent antisera are first bound to staphylococcal protein A. Soluble antigens were also identified specifically by the slide coagglutination test and by a sandwich immunofluorescence assay. The latter test may be useful in detecting antigen in body fluids of patients with legionellosis or in environmental samples.

Slide agglutination test for serogrouping Legionella pneumophila and atypical Legionella-like organisms.

A simple, inexpensive slide agglutination test correctly identified the serogroup to which 38 Legionella pneumophilia strains belonged, and distinguished them from two atypical, Legionella-like organisms.

Serological relationships of type I antigens of group B streptococci.

Some of the complex antigenic relationships of type I group B streptococci from various clinical sources were defined by means of immunodiffusion, absorption, and precipitin tests. Three predominant types are described: Ia, Ib, and Ii. Methods for preparing antisera for differentiating type I strains are presented.

Type-specific antigens of group B type Ic streptococci.

The type-specific antigens of group B type Ic (old designation type Ii) streptococci were extracted, purified, and characterized by serological and chemical methods. The Ia antigen, shared by types Ia and Ic, is a polysaccharide composed of 69% galactose and 25% glucosamine (i.e., 31% N-acetyl-glucosamine). However, these monosaccharides failed to inhibit significantly the quantitative precipitin reactions between purified antigen and type Ia antiserum. Indications are that the immunodominant group of this antigen consists of more than a simple monosaccharide. The Ic antigen, shared by types Ib and Ic, is a protein unrelated to the X and R protein antigens. Ic antigen consists of two serologically active determinants, one of which is susceptible to both trypsin and pepsin digestion and the other to pepsin but not to trypsin digestion. Acrylamide gel electrophoresis of the partially purified Ic antigen resulted in the occurrence of both determinants throughout the length of the gel, as shown by double gel diffusion slides.

Identification of group B streptococci by immunofluorescence staining.

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.

Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.

Comparison of slide agglutination test and direct immunofluorescence assay for identification of Legionella isolates.

It is technically impractical for many clinical laboratories to use the direct immunofluorescence assay for identifying and serogrouping clinical isolates of Legionella. We compared the results obtained with the direct immunofluorescence assay with the results of a simple and less-demanding slide agglutination test for identifying 15 serogroups representing seven Legionella species. The slide agglutination test was in complete agreement with the direct immunofluorescence assay, and the serogroup to which 64 clinical isolates of Legionella belonged was correctly identified. With polyvalent, pooled antisera and absorbed, serogroup-specific antisera, the slide agglutination test is a useful alternative to the direct immunofluorescence assay in the diagnosis of Legionella infections and for studying the serological relationships of Legionella-like organisms.