Biomedical subjects
Publications and source records attributed to H W Wilkinson.
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.
Isolates of Legionella pneumophila that are serologically different from strains of serogroups 1 through 5 were obtained from lung biopsy tissue or pleural fluid from three renal transplant recipients in Chicago, Ill. These strains were placed in a newly designated L. pneumophila serogroup, serogroup 6, on the basis of fluorescent-antibody staining characteristics. An L. pneumophila strain obtained from Bethesda, Md., one from Houston, Tex., and one from Oxford, England, also belong to this new serogroup. L. pneumophila serogroup 6 appears to be widely distributed geographically.
A group of related bacteria designated atypical Legionella-like organisms (ALLO) has been identified. ALLO, like L. pneumophila, are fastidious gram-negative rods that grow well on charcoal yeast extract (CYE) agar and produce ground glass colonies and browning of modified yeast extract agar. Unlike L. pneumophila, ALLO do not grow well on Feeley-Gorman (FG) agar, and on CYE agar they fluoresce under longwave ultraviolet light. ALLO and L. pneumophila have a similar predominance of branched-chain forms among total cellular fatty acids but have distinctive fatty-acid profiles. 2 patients with culture-verified ALLO pneumonia and 10 with pneumonia of uncertain aetiology who seroconverted to ALLO offer evidence that ALLO may be a cause of community-acquired pneumonia. Like L. pneumophila, ALLO appear to be water-associated; both persons with culture-verified ALLO infection were exposed to fresh water or its contents before becoming ill, and two strains of ALLO were isolated from water or wet environments.
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.
A crude extract of Escherichia coli O13:K92:H4 inhibited 97% of positive indirect immunofluorescence titers against a variety of gram-negative bacterial antigens while lowering Legionella pneumophila titers in only 6% of sera from patients with suspected legionellosis. Legionella-specific titers were the result of immunoglobulins G, M, and A, singly or in combination.
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.
Evidence obtained by others who used direct immunofluorescence staining to demonstrate serological differences among strains of Legionnaires disease bacterium prompted this study of parameters influencing the ability of the indirect immunofluorescence test to detect human antibodies to Legionnaires disease bacterium. A total of 25 Legionnaires disease bacterium strains, representing four serogroups, were used as immunofluorescence antigens to test selected human sera. The use of diethyl ether in preparing the antigens was discontinued when it was found that titers against ether-killed group 2 (Togus 1-like) antigens were impossible to determine. Instead, heat-killed suspensions of Legionnaires disease bacterium in 0.5% buffered normal chicken yolk sac were used to show the serogroup diversity of the strains and the serogroup specificity of the antibody response of some, but not all, patients with serological evidence of Legionnaires disease. These studies suggest that multiple antigens should be used in serological tests for Legionnaires disease. Furthermore, the fact that some sera contain antibodies that bind equally well to strains of all four serogroups implies that demonstration of a fourfold increase in titer of paired sera when tested with a single antigen should not be interpreted as evidence of infection with a strain of the same serogroup.
Differences in results of microagglutination tests for the Legionnaires' disease bacterium on untreated sera and sera treated with 2-mercaptoethanol suggest that, like other agglutination tests, the microagglutination test is heavily dependent on the presence of IgM antibody.
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.
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.
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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.
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.
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.
Within a six-day period in March, 1974, three infants born at a hospital in central Arkansas developed meningitis caused by group B, type III Streptococci. Three factors suggested nosocomial transmission of the organism in the nursery: (1) the three infants were born in a six-day period, (2) four weeks after their infants' births, none of the parents had positive cultures for group B streptococci, and (3) 31% of infants born in the hospital in March were colonized with group B, type III streptococci, while in April, after control measures in the nursery were instituted, only 2% of infants were colonized with this type (p less than 0.0002). Colonized infants were treated with penicillin, but follow-up cultures at two and six weeks showed that half the infants tested were still colonized. The number of personnel colonized with group B streptococci was not significantly different in personnel exposed to infants when compared with those that were not, and handwashing and environmental cultures were negative for group B streptococci. The results of this investigation give additional support to the concept that nosocomial transmission of group B streptococci can occur and may be effectively interrupted by control measures in the nursery.
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.
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.
The biochemical and serological characteristics of nonhemolytic group B streptococci of human, bovine, and ichthyic origin were described.