Antigenic analysis of Legionella pneumophila and Tatlockia micdadei (Legionella micdadei) by two-dimensional (crossed) immunoelectrophoresis.

The antigens of the six serogroups of Legionella pneumophila were compared by two-dimensional (crossed) immunoelectrophoresis by using rabbit antisera to serogroups 1, 2, 3 and 4. The close relationship among the serogroups was shown by the fact that 27 of the 31 antigens demonstrated so far were common. However, distinctive group-specific antigens with slow electrophoretic mobility were observed for serogroups 1, 2, 3, and 4. When intact serogroup 1 organisms were extracted with EDTA, the group-specific antigen was recovered in a virtually pure form. The group-specific antigen was pronase resistant, heat stable, and amphiphilic and had a surface location, all of which are properties suggestive of lipopolysaccharide. L. pneumophila shared four to five antigens with Tatlockia micdadei (Legionella micdadei). The large number of common antigens in the serogroups of L. pneumophila has important implications for the specific detection of antigens and antibodies by fluorescent and other tagged antibody methods.     

Acid-fast bacilli in sputum: a case of Legionella micdadei pneumonia.

Legionella micdadei has been implicated as a cause of nosocomial pneumonia. There are no reports of L. micdadei pneumonia diagnosed by acid-fast stain of expectorated sputum. We report a case of L. micdadei pneumonia in which expectorated sputum harbored acid-fast bacteria that reacted specifically with fluorescent antiserum to L. micdadei, confirmed by culture. In a patient at risk for nosocomial infection, the differential diagnosis of a positive sputum stain for acid-fast bacilli should include L. micdadei in addition to mycobacteria. Therapy for L. micdadei infection should be considered pending confirmation of the diagnosis.     

Retrospective examination of lung tissue specimens for the presence of Legionella organisms: comparison of an indirect fluorescent-antibody system with direct fluorescent-antibody testing

Although direct fluorescent-antibody (DFA) testing has been used successfully for a number of years to detect legionellae in clinical specimens, the number of known species and serogroups of Legionella has now increased to such an extent that the performance of DFA testing for all serological variants is impractical. Lung homogenates that were submitted to the Centers for Disease Control, Atlanta, Ga., from patients with suspected legionellosis, from November 1977 through May 1982, were originally screened by DFA testing. In our study 498 of these lung homogenates were screened by indirect fluorescent-antibody (IFA) testing, using a panvalent antiserum pool containing antibodies to 25 serological variants of Legionella spp. Fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin was used as the second antibody of the sandwich system. For positive homogenates, i.e., those containing Legionella organisms, species and serogroup identification was made by IFA staining with polyvalent serum pools and then with monovalent antiserum. Of the 498 homogenates screened, 39 (7.8%) were positive by IFA testing. Four (0.8% of total; 10.3% of positive homogenates) of these had previously been negative by DFA testing, but subsequent testing showed that they contained Legionella organisms for which DFA reagents were not available at the initial screening. All specimens that were positive by DFA testing were also positive by IFA testing. IFA testing with polyvalent antisera is a simple, efficient method which is at least as sensitive as DFA testing and which can be used by clinical laboratories to cope with the increasing number of known serological variants of Legionella spp.     

Natural killing of fibroblasts infected with low-passage clinical isolates of human cytomegalovirus.

Fibroblasts infected with most low-passage clinical isolates of human cytomegalovirus (CMV) were as susceptible to lysis by human natural killer (NK) cells as high passage AD-169-infected fibroblasts. NK lysis occurred despite the absence of detectable CMV-specific late membrane antigen(s) on the majority of the target cells infected with most of the low passage strains. The magnitude of NK lysis of different CMV-infected target cells did not correlate with their ability to induce IFN-alpha. NK cell-mediated lysis of cells infected with low-passage clinical isolates of CMV required both NK cells and HLA-DR+ accessory cells, as previously shown for AD-169-infected target cells.     

Polyamine biosynthesis in cells infected with different clinical isolates of human cytomegalovirus.

Previous work in this laboratory showed that polyamine biosynthesis was stimulated in fibroblasts following infection with the AD169 strain of human cytomegalovirus (HCMV) or with murine cytomegalovirus (MCMV) (Tyms et al: Biophysics Research Communications 86:312-318, 1979; Advances in Polyamine Research 4:507-517, 1983). Here we compare the affect of AD169 on polyamine production in infected fibroblasts with that of the unusual Colburn strain of HCMV. The Colburn virus is unusual in that it was isolated from a 7 year old boy with encephalitis and molecular studies indicated the virus was simian like (Huang et al: Journal of Virology 26:718-723, 1978). As a consequence of CMV infection a two to ten fold increase in the spermine content of fibroblast cells is observed. Radiolabel transfer experiments show that spermine is synthesized throughout virus infection. Indeed, spermidine and spermine are specifically incorporated into the purified virions of the AD169 and Colburn strains of HCMV. Furthermore, polyamine biosynthesis is stimulated in fibroblast cells infected with a number of low passage clinical isolates of HCMV. Inhibition of polyamine biosynthesis in HCMV infection may provide a specific and novel target for antiviral chemotherapy.     

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.     

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002-2006.

This study investigated the hypothesis that the genotype distribution of Legionella isolates from sporadic patients with Legionnaires' disease differs from that of Legionella strains in the environment. An amplified fragment-length polymorphism (AFLP) assay was used to genotype patient-derived and environmental Legionella isolates. The three Legionella pneumophila genotypes isolated most frequently from human respiratory secretions were AFLP types 004 Lyon, 010 London and 006 Copenhagen. These genotypes were cultured significantly less frequently from environmental samples (50% vs. 4%; p <0.001). The most frequently observed L. pneumophila serogroup 1 genotype among patient-derived isolates was 004 Lyon (32%). This genotype was cultured from only one of 6458 environmental samples. The positive sample contained 1.26 x 10(6) CFU/mL and originated from a whirlpool spa that had not been disinfected and had been maintained at 36 degrees C for several months. Overall, the distribution of genotypes differed significantly among patient and environmental isolates. A possible explanation is that virulent strains may exist in potential environmental sources at undetectable concentrations.     

Retrospective identification and characterization of Candida dubliniensis isolates among Candida albicans clinical laboratory isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected individuals

Fungal opportunistic infections, and in particular those caused by the various Candida species, have gained considerable significance as a cause of morbidity and, often, mortality. The newly described species Candida dubliniensis phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The present study was designed to determine the frequency at which this new species is not recognized in the clinical laboratory, to determine the patient populations with which C. dubliniensis is associated, to determine colonization versus infection frequency, and to assess fluconazole resistance. Over a 2-year period, 1,251 isolates that were initially identified as C. albicans by a hospital clinical laboratory were reevaluated for C. dubliniensis by inability to grow at 45 degrees C, colony color on CHROMagar Candida medium, coaggregation assay with Fusobacterium nucleatum, and sugar assimilation profiles (API 20C AUX yeast identification system). A total of 15 (1.2%) isolates from 12 patients were identified as C. dubliniensis. Ten of the patients were found to be immunocompromised (these included patients with human immunodeficiency virus infection or AIDS, cancer patients receiving chemotherapy, and patients awaiting transplantation). Thirteen isolates were highly susceptible to fluconazole (MIC, <0.5 microgram/ml). Three isolates from one patient, genotypically confirmed as the same strain, showed variable susceptibility to fluconazole. The first isolate was susceptible, whereas the other two isolates were dose-dependent susceptible (MIC, 16.0 microgram/ml). These data confirm the close association of C. dubliniensis with immunocompromised states and that increased fluconazole MICs may develop in vivo. This study emphasizes the importance of screening germ-tube-positive yeasts for the inability to grow at 45 degrees C followed by confirmatory tests in order to properly identify this species.     

Human papilloma viruses (HPV): characterization of four different isolates.

Out of 50 papilloma virus isolates from individual human warts (verrucae vulgares and verrucae plantares) 36 were analyzed for cleavage pattern of their DNA after restriction enzyme digestion with the endonucleases EcoRl, BamHI, Hind II, Hind III, Hpa II, and Hae III. Furthermore, the electrophoretic mobility of virion proteins from some of the same as well as from the additional isolates was studied by SDS-polyacrylamide gel electrophoresis. Four different cleavage patterns were observed: While three individual isolates (HPV 1, HPV 2, HPV 3) had many cleavage sites in common, and differed only in a few sites, a fourth one (HPV 4) was found to be entirely different. The electrophoretic mobility of proteins of HPV 4 was also observed to differ from those of HPV 1–3. cRNA transcribed either from HPV 1 or HPV 4 did not hybridize with the heterologous isolate. Rabbit antiserum reacting highly against HPV 1 did not react with HPV 4 proteins in complement fixation tests. We thus conclude that several human papilloma viruses exist: While HPV 1–3 are closely related isolates, HPV 4 represents a new human papilloma virus, profoundly different from the previous ones as far as base composition and antigenicity are concerned. Preliminary data suggest that the latter type occurs in approximately 20% of verrucae vulgares with low virus production, whereas HPV 1, representative for about 70% of these papillomas, predominates in warts with high virus yields.     

Phenotypic and molecular characterization of clinical isolates of Mycobacterium elephantis from human specimens

Eleven strains of a rapidly growing mycobacterium were isolated from patient specimens originating from various regions of the province of Ontario, Canada, over a 2-year period. Unique high-performance liquid chromatography (HPLC) and PCR-restriction enzyme pattern analysis (PRA) profiles initially suggested a new Mycobacterium species, while sequencing of the 16S rRNA gene revealed a sequence match with Mycobacterium sp. strain MCRO 17 (GenBank accession no. X93028), an isolate determined to be unique which is to date uncharacterized, and also a close similarity to M. elephantis (GenBank accession no. AJ010747), with six base pair variations. A complete biochemical profile of these isolates revealed a species of mycobacteria with phenotypic characteristics similar to those of M. flavescens. HPLC, PRA, and 16S rRNA sequencing of strain M. elephantis DSM 44368(T) and result comparisons with the clinical isolates revealed that these strains were in fact M. elephantis, a newly described species isolated from an elephant. All strains were isolated from human samples, 10 from sputum and 1 from an axillary lymph node.     

Genomic variants among human cytomegalovirus (HCMV) clinical isolates: the glycoprotein n (gN) paradigm

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, which are supposed to be implicated in strain-specific tissue tropism and HCMV-induced immunopathogenesis. One highly variable gene is ORF UL73, encoding for the envelope glycoprotein gN, which displays both a structural and an immunologic role as a component of the high-molecular weight complex gC-II. UL73 showed clustered polymorphisms, which originate four distinct genomic variants, denoted gN-1, gN-2, gN-3, and gN-4. This review reports the main features of gN genotypes and their potential implications on HCMV biologic properties. The clinical impact of gN variants is also discussed. This overview on gN clustered polymorphisms should be useful as a prototype model for a better understanding of the biologic and clinical relevance of HCMV clinical isolates genetic variability.     

Bronchus-associated lymphoid tissue (BALT) in human fetal and infant lung

Bronchus-associated lymphoid tissue (BALT) has been defined as the organized lymphoid tissue of the lung. Although well described in a variety of animal species, documentation of its presence and development in human lung is limited. Because the tissue to volume ratio in adult lungs is so low, a systematic search for BALT would involve so many sections as to be impractical. In this study, therefore, we have studied post-mortem specimens of fetal (n=102) and infant (n=17) lungs, which have a much higher tissue to volume ratio. Fetal death was due to various causes but all but two infants died from sudden infant death syndrome. In the fetal lungs, the presence of BALT was almost invariably associated with chorioamnionitis or intrauterine pneumonia, being present in 24 of 51 of these cases (47 per cent). The earliest ill-defined lymphoid aggregate was seen at 16 weeks' gestation, while lymphoepithelium, a hallmark of mucosaassociated lymphoid tissue, could be identified at 20 weeks. In 51 fetuses without infection, BALT was found in only five cases (10 per cent). BALT was identified in 13/17 (77 per cent) of infant lungs and well-developed lymphoepithelium was evident in four cases. This study shows that BALT may be present in the human fetal and infant lung, but that its appearance is probably dependent on antigenic stimulation.     

Phenotypic and molecular characterization of three clinical isolates of Mycobacterium interjectum.

Introduction of molecular biology-based technology into an Australian mycobacterial reference laboratory has resulted in the identification of three isolates of Mycobacterium interjectum in the past 12 months. Conventional phenotypic methods failed to identify the species of these isolates, and high-performance liquid chromatography found that only one of the three isolates had a mycolic acid pattern similar to that of the type strain. In contrast, all three isolates were rapidly identified as M. interjectum by 16S rRNA gene sequence analysis. Two isolates were recovered from the lymph nodes of children with cervical lymphadenitis, confirming the pathogenicity of this organism. However, the third isolate was obtained from the sputum of an elderly male with chronic lung disease without evidence of clinical or radiological progression, suggesting that isolation of M. interjectum should not imply disease. With the increasing use of molecular biology-based technology in mycobacterial laboratories, M. interjectum may be recognized more frequently as a pathogen or commensal organism.     

Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (CAA)

Pathogenicity for chicks of 11 isolates of chicken anaemia agent (CAA) isolated in Japan was examined. Each strain produced aplastic anaemia in 100% of chicks when inoculated at 1 day of age with mortalities ranging from 20 to 90%. Incidence of anaemia in chicks inoculated at 7 days of age varied from 0 to 87.5% according to the isolates, but no chick died. The susceptibility of chicks at various ages against the G1 (Gifu-1) and A2 strains was compared. Both strains produced anaemia in 100% of the chicks when inoculated at 1, 2, 3 or 4 days of age. Half of the chicks inoculated with the A2 strain at 8 days of age showed anaemia, but none of those inoculated with the G1 strain did. No chick showed anaemia from either strain when inoculated at 14 days of age. The antigenicities of the isolates were compared by cross neutralisation and indirect immunofluorescent antibody methods. No serological distinction was recognised among the isolates.     

Antiserum-agar plate method for simultaneous detection and direct isolation of Legionella species in clinical and environmental specimens.

Colonies of Legionella pneumophila serotypes 1 through 6, L. micdadei, L. bozemanii, L. dumoffii, and L. gormanii, which were developed on filtered yeast extract agar containing polyvalent antiserum, were surrounded by distinct, specific precipitin rings.     

Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia.

Two monoclonal antibodies which react specifically with cells infected by cytomegalovirus (CMV) are described. One antibody, 6-E3, reacts with a 72,000-dalton protein that appears early in infection and remains localized in the cell nucleus. The other antibody, 6-C5, reacts with an 80,000-dalton protein that appears late in infection and remains localized in cytoplasmic inclusion bodies. Both monoclonal antibodies react with conventional laboratory strains of CMV and can be used in immunofluorescence assays to identify clinical isolates of CMV in culture. Preliminary tests on lung tissues from patients with CMV pneumonia show that only antibody 6-C5 detects CMV infection in primary clinical specimens. A comparison of culture, histological, and immunological methods demonstrates that the monoclonal antibodies possess sufficient specificity and sensitivity to warrant their continued development as immunodiagnostic tools for the detection of CMV infection in both tissue culture and tissues obtained directly from patients.     

Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9.

The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae.     

Limited applicability of direct fluorescent-antibody testing for Bordetella sp. and Legionella sp. specimens for the clinical microbiology laboratory

The rapid diagnosis of infections with Bordetella and Legionella species is important for patient management. With observed increases in direct fluorescent-antibody (DFA) testing volumes, we retrospectively compared the performance characteristics of DFA testing to those of culture and PCR. For Bordetella sp., samples were classified as positive by DFA testing (184 [3%] of 6,195 samples) and culture (150 [2%] of 6,251 samples) significantly less often than by PCR (2,557 [10%] of 26,929 samples). Of 360 samples tested by both DFA and PCR methods, 81 (16 by DFA testing and 79 by PCR) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 18% and 99%, respectively. Of 1,426 samples tested by both DFA and culture methods, 48 (44 by DFA testing and 15 by culture) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 73% and 98%, respectively. For Legionella sp., samples were identified as positive by DFA testing (31 [0.25%] of 12,597 samples) and culture (85 [0.6%] of 13,572 samples) significantly less often than by PCR (27 [4%] of 716 samples). Of 62 samples tested by both DFA and PCR methods, none were positive for Legionella sp. by DFA testing and 3 were positive by PCR. Of 3,923 samples tested by both DFA and culture methods, 22 (3 by DFA testing and 21 by culture) were positive for Legionella sp., with a sensitivity and specificity of DFA testing of 9.5% and 100%. Overall, DFA testing for Bordetella sp. and Legionella sp. is an insensitive method, and despite its continued popularity, clinical microbiology laboratories should not offer it when more sensitive tests like PCR are available.     

Acetylene reduction (dinitrogen fixation) by clinical isolates of Klebsiella pneumoniae.

Freshly isolated clinical strains of Klebsiella were tested for the ability to fix dinitrogen by the acetylene reduction assay. Ability to detect this trait was markedly affected by cultural conditions. When the test was run at 37 degrees C in the presence of yeast extract (50 mg/liter), only 1.6% of the organisms were diazotrophs, whereas this temperature without yeast extract yielded 12.9% positive cultures. The optimum condition found was 28 degrees C without yeast extract (21.9% positive); therefore, search for diazotrophy in clinical strains should not be conducted at the usual incubation temperature. There was a high incidence of indole-positive strains among diazotrophs. No such correlation was noted with any other biochemical trait or antibiotic susceptibility tested. The significance of this correlation is not apparent.